Every-other-day Feeding Research Articles

Intermittent Fasting-Improved Glucose Homeostasis Is Not Entirely Dependent on Caloric Restriction in db/db Male Mice.

Intermittent fasting (IF), which involves prolonged fasting intervals accompanied by caloric restriction, is an effective dietary treatment for obesity and diabetes. Although IF offers many benefits, it is difficult to determine whether these benefits are the consequences of caloric restriction. Every-other-day feeding (EODF) is a commonly used IF research model. This study was designed to identify other effectors of EODF, in addition to caloric restriction, and the possible underlying mechanisms. Diabetic db/db mice were divided into three groups: ad libitum (AL), meal-feeding (MF) and EODF. The MF model was employed to attain a level of caloric restriction comparable to EODF, with food distribution evenly divided between 10 AM and 6 PM, thereby minimizing the fasting interval. EODF yielded greater improvements in glucose homeostasis than MF in db/db mice by reducing fasting glucose levels and enhancing glucose tolerance. However, these effects on glucose metabolism were less pronounced in lean mice. Furthermore, ubiquitination of the liverspecific glucocorticoid receptor (GR) facilitated its degradation, and downregulating Kruppel-like factor 9 (KLF9), which ultimately suppressed liver gluconeogenesis in diabetic EODF mice. Although GR and KLF9 might mediate the metabolic benefits of EODF, the potential benefits of EODF might be limited by elevated serum glucocorticoid (GC) levels in diabetic EODF mice. Overall, this study suggests that the metabolic benefits of EODF in improving glucose homeostasis are independent of caloric restriction, possibly due to the downstream effects of liver-specific GR degradation.

Every-other day (EOD) feeding regime decreases oxidative stress and inflammatory cascade in mouse liver: The immunohistochemical study

IntroductionPositive effects of calorie restrictions (CR) and EOD include decreased body weight, prolonged life span, but also changes in metabolism of the liver. In present paper our aim was to examine antioxidative enzymes: Catalase (CAT) and Manganese superoxidative dismutase (MnSOD, SOD2) and Glutathione peroxidase 4 (Gpx-4) in connection to caspase-3 and inflammatory mediators (IL-1β and TNF- α) in EOD liver tissue in comparison to control mice. MethodsAfter 9 months of EOD treatment male mouse liver tissue was harvested and prepared for analysis. Protein semi-quantitative estimation and cellular immunolocalization was performed for CAT, SOD2, Gpx-4, caspase-3, IL-1β and TNF- α in liver tissue of mice fed every-other day in comparison to control (AL fed) animals. ResultsAfter prolonged EOD feeding in mice we observed decreased level of SOD2 and Gpx-4, decreased caspase-3, IL-1β and TNF-α expression in liver tissue on protein level measured by semi-quantitative DAB staining intensity. ConclusionFor the first time we showed immunolocalization of major antioxidative enzymes (CAT, SOD2, Gpx-4) in liver tissue after DR. Decrease of two major antioxidant enzymes combined with decrease of apoptotic marker and inflammatory factors indicate decrease in oxidative stress as the result of fast in EOD feeding regime.

Estimation of Bone Trace Elements Following Prolonged Every-other Day Feeding in C57BL/6 Male and Female Mice

The purpose of this study was to examine the effect of prolonged every-other day (EOD) feeding on bone trace elements. Four-week old C57BL/6 female (n = 12) and male (n = 12) mice were employed in this experiment. Animals were assigned to four groups: ad libitum—AL (males and females), EOD fed (males, females). After 9 months, the mice were sacrificed. Long bones (humerus and radius) were isolated and prepared for analysis using inductively coupled plasma optical emission spectrometry to determine the Fe, Zn, Mo, Co, Cu, Mn, Cr contents. Estimation of cathepsin K expression on bone slides was performed to determine the activity of osteoclasts in bones of EOD- and AL-fed animals. Higher content of Fe in EOD-fed females compared to AL-fed females was found. In EOD-fed males, a significantly higher amount of Mo (p < 0.005) and Co (p < 0.05) in comparison to AL-fed males was noted. Gender differences in amounts of trace elements in control AL-fed males vs. females were observed. EOD feeding influences the amount of some trace elements in long bones of female and male C57BL/6 mice. However, this is not influenced by the activity of bone cells.

Every-other-day feeding exacerbates inflammation and neuronal deficits in 5XFAD mouse model of Alzheimer's disease

Food restriction has been widely associated with beneficial effects on brain aging and age-related neurodegenerative diseases such as Alzheimer's disease. However, previous studies on the effects of food restriction on aging- or pathology-related cognitive decline are controversial, emphasizing the importance of the type, onset and duration of food restriction. In the present study, we assessed the effects of preventive every-other-day (EOD) feeding regimen on neurodegenerative phenotype in 5XFAD transgenic mice, a commonly used mouse model of Alzheimer's disease.EOD feeding regimen was introduced to transgenic female mice at the age of 2 months and the effects on amyloid-β (Aβ) accumulation, gliosis, synaptic plasticity, and blood-brain barrier breakdown were analyzed in cortical tissue of 6-month-old animals. Surprisingly, significant increase of inflammation in the cortex of 5XFAD fed EOD mice was observed, reflected by the expression of microglial and astrocytic markers. This increase in reactivity and/or proliferation of glial cells was accompanied by an increase in proinflammatory cytokine TNF-α, p38 MAPK and EAAT2, and a decrease in GAD67. NMDA receptor subunit 2B, related to glutamate excitotoxicity, was increased in the cortex of 5XFAD-EOD mice indicating additional alterations in glutamatergic signaling. Furthermore, 4 months of EOD feeding regimen had led to synaptic plasticity proteins reduction and neuronal injury in 5XFAD mice. However, EOD feeding regimen did not affect Aβ load and blood-brain barrier permeability in the cortex of 5XFAD mice.Our results demonstrate that EOD feeding regimen exacerbates Alzheimer's disease-like neurodegenerative and neuroinflammatory changes irrespective of Aβ pathology in 5XFAD mice, suggesting that caution should be paid when using food restrictions in the prodromal phase of this neurodegenerative disease.

Every-Other-Day Feeding Decreases Glycolytic and Mitochondrial Energy-Producing Potentials in the Brain and Liver of Young Mice.

Intermittent fasting is used to reduce body mass in obese adult humans and animals. However, information on the impact of one type of intermittent fasting (IF) called every-other-day feeding (EODF) on young animals is scarce. In this study, 1-month-old mice of both sexes were subjected to a 4-week regimen of EODF using age-matched counterparts fed ad libitum as controls. At the end of EODF exposure, experimental male and female mice weighed 14 and 13% less than the control counterparts. The EODF regimen resulted in lower liver levels of glycogen, glucose, and lactate, but did not affect lactate level in mouse cerebral cortex of both sexes. Activities of key glycolytic enzymes (hexokinase, phosphofructokinase, and pyruvate kinase) in liver of experimental mice were lower than those in controls. In the cerebral cortex, only hexokinase and pyruvate kinase activities were lower than in controls, but phosphofructokinase activity was not affected in IF females and was higher in IF males as compared with ad libitum fed males. Mitochondria isolated from liver of IF mice had lower respiratory control ratios, but those from the cortex had the same values as control animals. The concentration of β-hydroxybutyrate and the activity of β-hydroxybutyrate dehydrogenase were lower in the IF mouse liver, but not changed or enhanced in the IF cerebral cortex. Thus, animal responses to IF do not depend significantly on sex and are directed to decrease energy metabolism to save resources, and the effects are more pronounced in the liver than in the brain.

The Effects of Age and Fasting Models on Blood Pressure, Insulin/Glucose Profile, and Expression of Longevity Proteins in Male Rats.

Intermittent fasting can be effective in reducing metabolic disorders and age-related diseases. However, there remain questions about the effects of fasting with respect to the age in which fasting begins, the fasting models, and the mechanisms involved. We investigated the effects of age of beginning fasting and chronic mild and severe fasting models on blood pressure (BP), insulin/glucose profile, and expression of klotho, sirtuin1 (SIRT1), and sirtuin3 (SIRT3) in male Wistar rats. Young (3 months), middle-aged (12 months), and old (22 months) animals were randomly divided into three subgroups and fed as ad libitum (AL), AL with fasting 1 day per week (FW), and AL with fasting every other day (EOD), respectively, for 3 months. The FW reduced the weight gain in young animals (p < 0.001 vs. AL), whereas EOD induced weight loss in all three age categories (p < 0.001). Aging was associated with high BP, high glucose, and insulin levels. Both FW and EOD feedings decreased BP and blood glucose level (p < 0.001) and EOD decreased insulin level (p < 0.05 vs. AL) in old animals. Parallel to aging, the expression of SIRT1 and klotho significantly decreased in plasma and EOD feeding recovered this defect. Both FW and EOD feedings increased the expression of SIRT3 in middle-aged and old rats. Age is a determining factor for the effectiveness of fasting and old animals respond more desirably to fasting. The effect of EOD fasting is more effective than FW fasting in improving the metabolic factors, partly through the recovery of SIRT1 and klotho.

Analysis of Bone Mineral Profile After Prolonged Every-Other-Day Feeding in C57BL/6J Male and Female Mice

Intermitted fasting or every-other-day feeding (EOD) has many positive effects in rodents and humans. Our goal was to describe how EOD influences bone mineral composition in female and male mice under prolonged EOD feeding. Male and female adult mice were fed EOD for 9 months. After this time, we used a direct method of measurement of mineral components in ashes of long bones (humerus and radius) to estimate the content of calcium (Ca), phosphorus (P), potassium (K), magnesium (Mg), and sodium (Na). We also performed histological analysis of sections of long bones. We found no significant changes in mineral composition between ad libitum and EOD fed males and females. We noted higher Ca and P contents in control males vs. females and lower content of Mg in control males vs. females. We observed the presence of marrow adipose tissue (MAT) in sections of EOD-fed females. EOD without supplementation during feeding days did not increase loss of mineral content of bones in C57BL/6J mice, but the presence of MAT only in EOD females indicates a gender-dependent response to EOD treatment in C57BL/6J mice.

SIRT1 Gain of Function Does Not Mimic or Enhance the Adaptations to Intermittent Fasting.

Caloric restriction (CR) has been shown to prevent the onset of insulin resistance and to delay age-related physiological decline in mammalian organisms. SIRT1, a NAD(+)-dependent deacetylase enzyme, has been suggested to mediate the adaptive responses to CR, leading to the speculation that SIRT1 activation could be therapeutically used as a CR-mimetic strategy. Here, we used a mouse model of moderate SIRT1 overexpression to test whether SIRT1 gain of function could mimic or boost the metabolic benefits induced by every-other-day feeding (EODF). Our results indicate that SIRT1 transgenesis does not affect the ability of EODF to decrease adiposity and improve insulin sensitivity. Transcriptomic analyses revealed that SIRT1 transgenesis and EODF promote very distinct adaptations in individual tissues, some of which can be even be metabolically opposite, as in brown adipose tissue. Therefore, whereas SIRT1 overexpression and CR both improve glucose metabolism and insulin sensitivity, the etiologies of these benefits are largely different.

Long-term intermittent feeding restores impaired GR signaling in the hippocampus of aged rat

Diminished glucocorticoid signaling is associated with an age-related decline in hippocampal functioning. In this study we demonstrate the effect of intermittent, every other day (EOD) feeding on the glucocorticoid hormone/glucocorticoid receptor (GR) system in the hippocampus of middle-aged (18-month-old) and aged (24-month-old) Wistar rats. In aged ad libitum-fed rats, a decrease in the level of total GR and GR phosphorylated at Ser232 (pGR) was detected. Conversely, aged rats subjected to EOD feeding, starting from 6 months of age, showed an increase in GR and pGR levels and a higher content of hippocampal corticosterone. Furthermore, prominent nuclear staining of pGR was observed in CA1 pyramidal and DG granule neurons of aged EOD-fed rats. These changes were accompanied by increased Sgk-1 and decreased GFAP transcription, pointing to upregulated transcriptional activity of GR. EOD feeding also induced an increase in the expression of the mineralocorticoid receptor. Our results reveal that intermittent feeding restores impaired GR signaling in the hippocampus of aged animals by inducing rather than by stabilizing GR signaling during aging.

Metabolic adaptations to short-term every-other-day feeding in long-living Ames dwarf mice

Restrictive dietary interventions exert significant beneficial physiological effects in terms of aging and age-related disease in many species. Every other day feeding (EOD) has been utilized in aging research and shown to mimic many of the positive outcomes consequent with dietary restriction. This study employed long living Ames dwarf mice subjected to EOD feeding to examine the adaptations of the oxidative phosphorylation and antioxidative defense systems to this feeding regimen. Every other day feeding lowered liver glutathione (GSH) concentrations in dwarf and wild type (WT) mice but altered GSH biosynthesis and degradation in WT mice only. The activities of liver OXPHOS enzymes and corresponding proteins declined in WT mice fed EOD while in dwarf animals, the levels were maintained or increased with this feeding regimen. Antioxidative enzymes were differentially affected depending on the tissue, whether proliferative or post-mitotic. Gene expression of components of liver methionine metabolism remained elevated in dwarf mice when compared to WT mice as previously reported however, enzymes responsible for recycling homocysteine to methionine were elevated in both genotypes in response to EOD feeding. The data suggest that the differences in anabolic hormone levels likely affect the sensitivity of long living and control mice to this dietary regimen, with dwarf mice exhibiting fewer responses in comparison to WT mice. These results provide further evidence that dwarf mice may be better protected against metabolic and environmental perturbations which may in turn, contribute to their extended longevity.

Muscle Physiology Changes Induced by Every Other Day Feeding and Endurance Exercise in Mice: Effects on Physical Performance

Every other day feeding (EOD) and exercise induce changes in cell metabolism. The aim of the present work was to know if both EOD and exercise produce similar effects on physical capacity, studying their physiological, biochemical and metabolic effects on muscle. Male OF-1 mice were fed either ad libitum (AL) or under EOD. After 18 weeks under EOD, animals were also trained by using a treadmill for another 6 weeks and then analyzed for physical activity. Both, EOD and endurance exercise increased the resistance of animals to extenuating activity and improved motor coordination. Among the groups that showed the highest performance, AL and EOD trained animals, ALT and EODT respectively, only the EODT group was able to increase glucose and triglycerides levels in plasma after extenuating exercise. No high effects on mitochondrial respiratory chain activities or protein levels neither on coenzyme Q levels were found in gastrocnemius muscle. However, exercise and EOD did increase β-oxidation activity in this muscle accompanied by increased CD36 levels in animals fed under EOD and by changes in shape and localization of mitochondria in muscle fibers. Furthermore, EOD and training decreased muscle damage after strenuous exercise. EOD also reduced the levels of lipid peroxidation in muscle. Our results indicate that EOD improves muscle performance and resistance by increasing lipid catabolism in muscle mitochondria at the same time that prevents lipid peroxidation and muscle damage.

Resveratrol Delays Age-Related Deterioration and Mimics Transcriptional Aspects of Dietary Restriction without Extending Life Span

A small molecule that safely mimics the ability of dietary restriction (DR) to delay age-related diseases in laboratory animals is greatly sought after. We and others have shown that resveratrol mimics effects of DR in lower organisms. In mice, we find that resveratrol induces gene expression patterns in multiple tissues that parallel those induced by DR and every-other-day feeding. Moreover, resveratrol-fed elderly mice show a marked reduction in signs of aging, including reduced albuminuria, decreased inflammation, and apoptosis in the vascular endothelium, increased aortic elasticity, greater motor coordination, reduced cataract formation, and preserved bone mineral density. However, mice fed a standard diet did not live longer when treated with resveratrol beginning at 12 months of age. Our findings indicate that resveratrol treatment has a range of beneficial effects in mice but does not increase the longevity of ad libitum-fed animals when started midlife.

Effects of Every-Other-Day Feeding on Prolactin Regulatory Mechanism in Transgenic Human Growth Hormone Mice

Transgenic mice overexpressing human growth hormone (hGH) exhibit accelerated aging with functional hyperprolactinemia and greatly depressed endogenous prolactin. Calorie restriction (CR) is widely recognized as the most effective experimental intervention to delay aging. The aim of the present work was to analyze the effects of lifelong overexpression of hGH on prolactin-gene expression as well as the dopamine production at the pituitary level and discern whether this mechanism changes as a function of feeding patterns. Ten-month-old mice fed every other day (EOD) were killed after one day of fasting. The results confirmed typical phenotypic features of these transgenic mice: an increase in body weight, very high hGH plasma concentrations, and hyperinsulinemia. There was a marked inhibition of the expression of the prolactin gene, together with an increased tyrosine hydroxylase (TH) and the long isoform of dopamine receptor type 2 (D2LR) gene expression at the pituitary level. These parameters were not affected by the EOD feeding pattern. These data may suggest an autocrine or paracrine effect of dopamine at the hypophyseal level on prolactin secretion that is independent of the feeding pattern.

Effect of every other day feeding diet on gene expression in normal and in long-lived Ames dwarf mice

Ames dwarf mutant mice are long-lived, hypoinsulinemic and hypoglycemic and exhibit enhanced sensitivity to injected insulin. Their phenotypic characteristics show many similarities to animals subjected to caloric restriction (CR) but Ames dwarf mice are not CR mimetics. Reducing daily food intake by 30% prolongs longevity in both normal and Ames dwarf mice. In the present study, the animals were subjected to a different type of CR, every other day feeding (EOD). Using real-time PCR, we have examined the expression of genes related to insulin signaling in the liver of normal and dwarf mice after 9 months of EOD. The results indicate that EOD produces some changes in the insulin and IGF1 signaling pathways, and that these changes are consistent with EOD increasing insulin sensitivity.

Enhancement of amphetamine-induced locomotor response in rats on different regimens of diet restriction and 2-deoxy- d-glucose treatment

Diet restriction (DR) in rodents increases lifespan, reduces age-related disease and pathology, increases stress responses, and maintains better function later into life compared with conventional ad libitum (AL) feeding. We have been investigating different DR regimens and also DR mimetics that stimulate stress response pathways that are activated by DR. By inhibiting glycolysis, feeding or injection of 2-deoxy- d-glucose (2DG) has been proposed as a DR mimetic and has been shown to provide neuroprotection. In the current study, we examined whether 2DG treatment produces behavioral changes similar to those observed in DR rats following stimulation of the dopaminergic (DA) system by d-amphetamine (AMPH). Male Fischer 344 rats were maintained on different dietary regimens: 40% daily DR (40% DR); every-other-day feeding (EOD); or AL with some groups provided food containing 0.4% 2DG or injected i.p. with 2DG. In addition, we examined the persistence of effects of DR or 2DG feeding after switching rats to AL. When locomotor activity was assessed at different time points following initiation of dietary treatments, we noted that the enhancement of AMPH-induced locomotor responses emerged earlier in DR rats than observed in 2DG fed rats, but 40% DR and EOD rats responded in a similar manner. Enhanced locomotor responses persisted in 2DG fed rats even when returned to normal diet for 1 month and in the case of DR rats even after 2 months of AL feeding. Three weeks of 2DG injections also enhanced AMPH response, but this effect was transient. The most important finding was that 2DG did not affect body weight or diet intake yet had effects similar to DR. Thus, 2DG appears to activate DA pathways in the same direction as DR does but without the necessity of reducing caloric intake.

Effects of Dietary Protein Concentration on Production Characteristics of Pond-Raised Channel Catfish Fed Once Daily or Once Every other Day to Satiation

We evaluated the effects of dietary protein and feeding regime on the production characteristics, processing yield, and body composition of channel catfish Ictalurus punctatus. Stocker-size fish with an average weight of 93 g/fish were stocked into twenty-four 0.4-ha ponds at a rate of 11,115 fish/ha. Fish were fed once daily or every other day (EOD) to apparent satiation with three commercial diets containing 28, 32, or 35% protein in a 2 × 3 factorial arrangement for 110 d. Compared with daily feeding, EOD feeding resulted in lower average net production, feed consumption, weight gain per fish, and feed conversion ratio and produced fewer marketable fish. The EOD ponds required less aeration than ponds with fish fed daily. Fish fed EOD had lower visceral fat and fillet fat, carcass yield, and fillet yield compared with fish fed daily. Dietary protein levels had no effect on net production, aeration time, processing yield, fillet composition, and feed consumption and weight gain per fish. However, the feed conversion ratio was higher for fish fed the 28% protein diet than for fish fed the other two diets, which was unexpected because we have not observed this in other studies. Neither dietary protein nor feeding regime had an effect on pond water total ammonia nitrogen, un-ionized ammonia, or nitrite-nitrogen concentrations; these nitrogenous compounds were highly variable among ponds and time. The results from the present study demonstrated that dietary protein levels in the range of 28% to 35% had no effect on channel catfish growth or production whether the fish were fed once daily or once EOD to satiation. Feeding EOD may improve feed efficiency and reduce feed cost and aeration time but will also reduce production and processing yield and extend the production cycle compared with feeding daily.

The effect of caloric restriction on the lateral diffusion constant of hepatocyte membrane proteins in C57BL/6 male mice of different ages: FRAP studies on liver smears

The lateral mobility of proteins in hepatocyte plasma membranes was compared in calorically restricted and ad libitum (AL)-fed C57BL/6 male mice in age groups from 7 to 28 months. Caloric restriction was achieved by means of the every-other-day (EOD) feeding regimen, maintained for various periods from 1 to 15 months. Protein lateral diffusion constant (D) in hepatocyte membranes was measured by means of fluorescence recovery after photobleaching (FRAP) in liver smears. The peroxide-induced autofluorescence (PIAF) was utilized as a fluorescent label. A mild (1 mM for 10 min) H 2O 2 treatment of liver smears produces oxidation of riboflavin that is bound to all proteins of the cell membrane. Using this technique, the average lateral diffusion constant (D) and the fractional recovery (FR) of these proteins can be measured. EOD feeding resulted in a significant decrease in body weights and also a significant increase in the values of D in all age groups after 1 month of EOD feeding. After 3.5 months of EOD feeding a further increase of D was observed (up to about 15%). Nevertheless no further change in D occurred if the EOD feeding was maintained for 6.5 or even 15 months. The negative linear age correlation of D observed in the AL-fed animals was present also under the EOD feeding; however, the whole regression equation shifted towards higher values. These experiments indicate that caloric restriction influences the lateral diffusion constant of membrane proteins in hepatocytes. The results are interpreted as a result of an increased protein turnover caused by the caloric restriction.

Dietary restriction suppresses age-related changes in dendritic spines

The effects of dietary restriction by every-other-day (EOD) feeding on dendritic spines in the aging rat neocortex were evaluated in Golgi preparations. After weaning, male Wistar rats were offered a 24% protein diet either ad lib (AL) or EOD. AL-fed groups were sacrificed at 6 and 24–25 months of age. EOD-fed groups were sacrificed at 6, 24, and 30 months. To assess the effects of EOD feeding late in life, another group was fed AL for 19 months, then EOD for 5 months and sacrificed when 24 months old. Spine density and configuration were quantified along 20 μm terminal tip segments from the basilar tree of layer V pyramidal cells of the parietal cortex. Evaluation of spine densities from the 6 and 24 months AL-fed groups showed that there was a significant loss of spines with normal aging (−38%). In EOD-fed rats, spine density did not differ significantly from AL age-matched controls at either 6 or 24 months of age. However, spine densities in 24-month-old rats diet restricted late in life and EOD-fed 30-month-old rats were the same as 6-month-old AL-fed controls and EOD 6-month-old rats, an observation suggesting protection of dendritic spines from age-related loss. Spines were categorized as either L-type (lollipop-shaped), which are more prevalent in young adults, or N-type (nubbin). With normal aging (comparing 6- and 24-month-old AL-fed groups) there was a significant decrease in L-type spines. However, all dietarily restricted groups showed retention of L-type spines. The results indicate that age differences in neuronal morphology may be attenuated by chronic dietary restriction initiated after weaning or late in life.

Effects of long-term restricted feeding on radial maze performance by aged rats

Rats were fed every other day (EOD) from 3–21 months of age prior to training in an 8-arm radial maze with retention intervals of 0, 1 or 5 hr imposed between the fourth and fifth choices. Their performance was compared to that of another aged group fed ad lib throughout life prior to radial maze training and to 3 month old controls. Rats fed ad lib until training adapted to the maze more slowly than young controls and were impaired in acquiring accurate spatial memory. EOD feeding eliminated age-related differences in the initial rate of adaptation to the radial maze but had no effect on the development of accurate spatial memory at any of the retention intervals. These results indicate that differences in deprivation history are probably not responsible for previous findings that radial maze training early in life prevents the appearance of age-related deficits in accurate spatial memory.

Effects of intermittent feeding on neurochemical markers in aging rat brain

Male Wistar rats were fed a diet of 24% protein either ad lib (AL) or every other day (EOD) beginning shortly after weaning. Rats were killed at 6 or 24-months of age. The corpus striatum, cerebral cortex, hippocampus, and cerebellum were assayed for muscarinic cholinergic receptor binding and the activities of choline acetyltransferase (ChAT), L-glutamic acid decarboxylase (GAD) and tyrosine hydroxylase (TH). Sensecent rats maintained on EOD feeding schedules from weaning showed a markedly higher density of striatal muscarinic binding sites and higher activities of cerebellar, hippocampal, and striatal ChAT, as compared with values in AL-fed rats. Regional GAD and TH activities were relatively unaffected by EOD feeding; however, at 6 months, EOD-fed rats had higher cortical GAD and lower cortical TH activities than corresponding AL-fed rats. EOD feeding for only 2 weeks produced no significant effects on neurochemical parameters examined in 24-month-old rats. We conclude that EOD feeding from weaning can alter neurochemical markers of senescence in rat brain; that cholinergic systems are affected in particular; and that the observed alterations produced by EOD feeding represent chronic rather than acute effects.