Age-dependent changes in glutamate oxidation by non-synaptic and synaptic mitochondria from rat brain

The oxidation of glutamate by non-synaptic and synaptic mitochondria from brains of 3-, 12- and 24-month-old rats was studied. With glutamate plus malate as substrates, non-synaptic mitochondria showed higher respiration rates than synaptic mitochondria in all the three age groups studied. The rate of oxidation of L-[1-¹⁴C] glutamate and the activities of NAD-glutamate dehydrogenase and aspartate aminotransferase were also higher in non-synaptic mitochondria compared with synaptic mitochondria in three age groups. With glutamate plus malate as substrates, a significant reduction in state 3 respiration was observed in both mitochondrial populations from 12- and 24-month-old rats compared with 3-month-old animals. Although an age-dependent decrease in the oxidation of L-[1-¹⁴C] glutamate was observed in both non-synaptic and synaptic mitochondria from aging rats, the oxidation of [1-¹⁴C]-2-oxoglutarate was unaltered in non-synaptic and synaptic mitochondria from senescent rats. The activity of NAD-glutamate dehydrogenase was decreased with age in both mitochondrial populations, whereas aspartate aminotransferase was not altered with age. The results indicate that the oxidation rate of glutamate in rat brain mitochondria is decreased during aging.     

Age-dependent changes in rat brain mitochondria of synaptic and non-synaptic origins

Synaptic and non-synaptic mitochondria were isolated from the brains of 3- 12-and 28-30-month-old female Fisher 344 rats. Total oxygen consumption and oxygen consumption due to mitochondrial respiration decreases 83% with increasing age in synaptic mitochondria using malate plus glutamate as substrate, but only 33% in non-synaptic mitochondria; succinate-driven activity is not affected. Succinate-driven superoxide generation decreases over 90% in both fractions; malate plus glutamate-driven superoxide generation decreases 50% in synaptic mitochondria only. The amount of c- and a-type cytochromes decreases approximately 50% in synaptic mitochondria. The absorbance wavelength maximum of cytochrome b decreases 2.6 nm in synaptic mitochondria from senescent brains but only 1.6 nm in non-synaptic mitochondria.     

Modification of mitochondrial respiration by aging and dietary restriction

Effects of aging and of dietary restriction on mitochondrial recovery and respiratory capacities have been assessed in mice. Old mice (23-26 months) did not differ from adult mice (9-12 months) in amounts of protein recovered in mitochondrial fractions of liver, brain and spleen, but did show a decline in specific activity of cytochrome c oxidase (cyt. c ox.) in liver and spleen. Age effects on in vitro respiration by mitochondria occurred in liver and spleen. In liver, only one substrate (beta-hydroxybutyrate) of four tested was respired at a different rate by old than by young mitochondria. Depression of state 3 respiration and 2,4-dinitrophenol (DNP)-uncoupled rates was observed for this substrate; however, this effect depended on expressing respiration on the basis of mitochondrial protein and was less overt if data were expressed per unit of cyt. c ox. activity. Old spleen mitochondria exhibited a grosser defect, showing a 40% decrease in the respiratory control index (RCI) for (succinate + rotenone)- supported respiration (the only substrate tested) due to a possible increase in state 4 rates. Effects of dietary restriction were assessed in liver and brain of 3-7-month-old mice underfed since weaning. Dietary restriction reduced recovery of total liver mitochondrial protein and liver cyt. c ox. specific activity. Liver mitochondria from restricted mice generally showed increased state 3 rates with no differences from controls in state 4 rates for respiration supported by glutamate or pyruvate + malate, resulting in an increased RCI for these substrates. DNP-uncoupled rates were also raised by dietary restriction. Unlike effects observed in old versus young mice, these differences obtained whether the data were expressed on the basis of mitochondrial protein or on cyt. c ox. activity. Electron microscopy of liver mitochondrial preparations revealed more non-mitochondrial contaminants in old mice and larger mitochondria in dietarily restricted mice. These findings are compatible with reports of age-dependent losses of liver mitochondria and suggest that dietary restriction may retard this loss.     

Effect of age on kinetics and carbon monoxide binding to cytochrome oxidase in synaptic and non-synaptic brain mitochondria

The kinetic parameters of cytochrome oxidase activity in synaptic and non-synaptic brain mitochondria from 3- and 30-month-old rats were determined at room temperature. The value of Km for cytochrome c increased only 12-13% with age. The maximal velocity did not change with age, but the value of Vmax in synaptic mitochondria is twice that observed in non-synaptic mitochondrial cytochrome oxidase. The kinetics of CO binding to cytochrome oxidase at temperatures from 183 to 225K were also studied in synaptic and non-synaptic mitochondria from 3- and 30-month-old rat forebrains. Age-dependent differences were observed only in mitochondria of synaptic origin. Following flash photolysis at low temperatures, CO migration to the iron requires crossing two free energy barriers separating two intermediate regions from the iron. In 3-month-old synaptic mitochondria, CO must migrate across a 10.3 kcal/mol barrier separating two intermediate regions, I2 and I; a 4.7 kcal/mol barrier separates the innermost region I from the iron. Each intermediate region in 3-month-old cytochrome oxidase can hold only one CO molecule. In 30-month-old synaptic mitochondria, 10.3 kcal/mol barriers separate the two intermediate regions as well as region I and the iron; each intermediate region can hold two CO molecules. Region I2 in non-synaptic cytochrome oxidase at either age can hold two CO molecules and the innermost region I holds only one CO molecule; energy barriers of approximately 10.3 kcal/moIe separate regions I2, I, and the iron. These age-dependent changes may reflect age-dependent conformational changes in cytochrome oxidase.     

Impairment of glutamate uptake and absence of alterations in the energy-transducing ability of old rat brain mitochondria

The proton electrochemical gradient has been measured in old brain mitochondria isolated from 2- or 24-month-old rats with the use of different respiratory substrates. With succinate as substrate, neither the respiratory rate, membrane potential or delta pH varied with age, indicating that the dielectric strength of the mitochondrial membrane was unaltered in old animals. The ohmic behavior of the membrane was tested in experiments in which the respiratory rate was partially inhibited by malonate, and was found to be unchanged with age. When glutamate plus malate were used as substrates, the respiratory rate was substantially reduced, and a drastic decrease in glutamate uptake was observed in old rat brain mitochondria.     

Effect of prolonged undernutrition on rat diaphragm mitochondrial respiration.

Previous studies have shown that undernutrition induces an impairment of the respiratory muscle function in patients with chronic lung disease. To explain this, we hypothesized that undernutrition could decrease oxidative metabolism in the diaphragm. We therefore examined the effect of prolonged undernutrition on diaphragm mitochondrial oxygen uptake with pyruvate and palmitate as substrates in adult rats. Ten rats served as controls (CTL). Ten nutritionally deprived rats (ND) received 40% of their estimated daily nutrition. Five weeks of undernutrition induced a 33% decrease in state 3 respiration with pyruvate plus malate as substrate (993 +/- 171 versus 1488 +/- 167 nmol atomic O/mg/min, P < 0.01) and a 39% decrease with palmitate plus malate (516 +/- 89 versus 850 +/- 165 nmol atomic O/mg/min, P < 0.05). With succinate plus rotenone, there was no significant difference in the respiratory rate between groups. In the ND group, we found a significant decrease in citrate synthase activity (P < 0.01), and also in reduced nicotinamine adenine dinucleotide (NADH) dehydrogenase activity (P < 0.05), which cannot alone induce such a state 3 respiratory decrease. This showed that undernutrition in rat diaphragm does not induce an alteration in protein complexes I, II, III, and IV, or the F complex containing the mitochondrial ATPase of the electron transport chain. In conclusion, the main result of this study was that prolonged undernutrition induced a decrease in mitochondrial respiration secondary to a significant reduction in NADH generation by the Krebs cycle, which may affect respiratory muscle function with implications for patient care.     

Exercise-induced reversal of age-related declines of oxidative reactions, mitochondrial yield, and flavins in skeletal muscle of the rat

The ability of gastrocnemius muscle homogenates to catalyze the oxidation of succinate, glutamate + malate, pyruvate + malate, palmitoyl-coenzyme A, decanoylcarnitine and palmitoylcarnitine in the presence of ADP decreased by approximately 32% in sedentary male Sprague-Dawley rats between the ages of 9 and 25 months. Following 21 weeks of treadmill training (running), such homogenates from 25-month-old animals catalyzed oxidations 55% more rapidly than those from 25-month-old sedentary rats, and 17% faster than those from 9-month-old sedentary rats. Total and peptide-bound flavin of gastrocnemius muscles also declined between 9 and 25 months of age and were elevated in the 25-month-old endurance trained rats to levels greater than both 9- and 25-month-old sedentary animals. The yield of protein in the mitochondrial fraction from the quadriceps femoris muscle decreased between 9 and 25 months and was restored to the 9-month level by endurance training. The kinetic characteristics of the isolated mitochondria were not influenced by age or exercise. These data indicate that 2-year-old rats retain the capacity to increase skeletal muscle oxidative capacity and mitochondrial population density in response to endurance training.     

Inhibition of mitochondrial NADH-ubiquinone oxidoreductase activity and ATP synthesis by tetrahydroisoquinoline

Effects of tetrahydroisoquinoline (TIQ) on mitochondrial respiration, NADH-ubiquinone oxidoreductase (complex I) activity and on adenosine triphosphate (ATP) synthesis were studied using mitochondria prepared from mouse brains. Tetrahydroisoquinoline significantly inhibited mitochondrial respiration supported by glutamate + malate, pyruvate + malate or alpha-ketoglutarate. Activity of complex I and synthesis of ATP were also significantly inhibited by TIQ. Mitochondrial respiration supported by succinate and subsequent ATP synthesis were not inhibited at all by 5 mM of TIQ. Our study has revealed a novel action of TIQ, which has been proposed as a candidate for an endogenous substance that may induce Parkinson's disease.     

Changes in intestinal glucose transport over the lifespan of the rat

Age-related changes in intestinal glucose absorption were studied using everted intestinal sacs and brush border membrane vesicles prepared from male F344 rats. Glucose uptake by everted intestinal sacs was greatest in young (2-3-month-old) as compared with adult (12-14-month-old) and old (24-month-old) rats. The greatest decrease in glucose uptake occurred between 2 and 12 months. The addition of phloridzin reduced glucose uptake to similar levels in all age groups, suggesting that the age-related change was in the carrier-mediated component of glucose transport. In order to localize the site of decreased carrier-mediated glucose transport, experiments were performed using brush border membrane vesicles. Vesicular glucose uptake in the presence of Na was significantly greater in vesicles prepared from 2-month-old rats (133 +/- 18 pmol/mg/s), compared with those prepared from 12-month-old rats (82 +/- 13 pmol/mg/s). Kinetic studies performed under non-equilibrium conditions demonstrated that the major effect of age was on the Na-dependent component of the brush border transport system. There was a reduction in the Vmax from 335 +/- 37 pmol/mg/s in the young to 217 +/- 22 pmol/mg/s in the adult, but there was no change in the Km. Isotope exchange studies performed under equilibrium conditions confirmed a decrease in the activity of the glucose transporter with age. No age-related changes in Na uptake by brush border membrane vesicles were observed. These findings suggest that a decrease in the number and/or activity of Na-linked glucose carriers may account for the decrease in intestinal glucose transport with age.     

The effect of gossypol and Lonidamine on electron transport in Ehrlich ascites tumor mitochondria

The effect of the association of gossypol and Lonidamine on the electron transport in Ehrlich ascites tumor mitochondria has been investigated by addition of drugs to isolated mitochondria. The results may be summarized as follows. (1) Low concentrations of gossypol increase the rate of oxygen consumption at the level of three energy-conserving sites of the respiratory chain. Higher concentrations result in an inhibition of oxygen consumption at (or near) both energy-conserving sites 1 and 2, while energy-conserving site 3 is unaffected. (2) Gossypol, at concentrations at which it exerts its uncoupling effect, stimulates ATPase activity. Higher concentrations inhibit the enzyme activity. (3) The addition of gossypol to mitochondria respiring on pyruvate plus malate or succinate induces a more oxidized state of NAD+ and cytochrome b, respectively. (4) Gossypol enhances the effect of Lonidamine on oxygen consumption. Lonidamine does not affect state 4 respiration, but in the presence of gossypol, it determines a marked decrease in the rate of oxygen consumption. The inhibition of oxidation of NAD-linked substrates is greater than that of FAD-linked substrates. (5) It may be concluded that gossypol is very effective in potentiating the effect of Lonidamine. Moreover, it may be suggested that the antitumor activity of Lonidamine is enhanced if it is used in combination with other drugs and/or treatments, such as hyperthermia, which modify the energy status of mitochondria.     

Lactate dehydrogenase is not a mitochondrial enzyme in human and mouse vastus lateralis muscle

The presence of lactate dehydrogenase in skeletal muscle mitochondria was investigated to clarify whether lactate is a possible substrate for mitochondrial respiration. Mitochondria were prepared from 100 mg samples of human and mouse vastus lateralis muscle. All fractions from the preparation procedure were assayed for marker enzymes and lactate dehydrogenase (LDH). The mitochondrial fraction contained no LDH activity (detection limit ∼0.05 % of the tissue activity) and the distribution of LDH activity among the fractions paralleled that of pyruvate kinase, i.e. LDH was fractionated as a cytoplasmic enzyme. Respiratory experiments with the mitochondrial fraction also indicated the absence of LDH. Lactate did not cause respiration, nor did it affect the respiration of pyruvate + malate. The major part of the native cytochrome c was retained in the isolated mitochondria, which, furthermore, showed high specific rates of state 3 respiration. This excluded artificial loss from the mitochondria of all activity of a possible LDH. It was concluded that skeletal muscle mitochondria are devoid of LDH and unable to metabolize lactate.     

Decrease of phosphorylating oxidation and increase of heat producing NADH oxidation in rat liver mitochondria during life-span prolongation of rats by calorie-restricted diet.

The influence of calorie-restricted diet, initiated at weaning, on some of the oxidative processes in liver homogenates and isolated mitochondria of 2-, 3-, 4-, 24-, 35- and 45-month-old male Wistar rats was studied in comparison with control ad libitum-fed 1-2 day-old rats and 0.5-, 1-, 2-, 3-, 4- and 24-month-old rats. It was shown that a calorie-restricted diet (at 37% of the ad libitum calorific level) did not change the rate of succinate oxidation coupled with oxidative phosphorylation in homogenates, but resulted in a decrease of succinate, glutamate plus malate and beta-hydroxybutyrate oxidation and cytochrome c-oxidase activity in isolated mitochondria without any uncoupling of oxidative phosphorylation or change in cytochrome content in the mitochondria. On the other hand, a significant increase in mitochondrial rotenone-insensitive NADH oxidation and a higher liver mass/body mass ratio in rats under the calorie-restricted diet was established. It may be considered that the activation of a heat-producing mechanism is a very important physiological function in such a condition.     

Treatment with dehydroepiandrosterone (DHEA) stimulates oxidative energy metabolism in the cerebral mitochondria. A comparative study of effects in old and young adult rats

The content of the neurosteroids, dehydroepiandrosterone (DHEA) in the brain decreases with aging. Also the oxidative energy metabolism is known to decrease with aging. Hence we examined the effects of treatment with DHEA (0.2 or 1.0 mg/kg body weight for 7 days) on oxidative energy metabolism in brain mitochondria from old and young adult rats. State 3 respiration rates in brain mitochondria from old animals were considerably lower than those in young adults. Treatment with DHEA stimulated state 3 and state 4 respiration rates in both the groups of the animals in a dose-dependent manner. In the old rats following DHEA treatment, the state 3 respiration rates became comparable to or increased beyond those of untreated young adults. In contrast to the old rats, stimulatory effect of DHEA treatment was of greater magnitude in the young adults. However, at higher dose (1.0 mg) the effect declined. Cytochrome aa3 content in the brain mitochondria from old rats was significantly low but the content of cytochrome b was unchanged while the content of cytochromes c+c1 had increased. Treatment with DHEA increased the content of cytochrome aa3 and b in old as well as in young adult animals. Higher dose of DHEA (1.0 mg) had adverse effect on the content of cytochrome c+c1. DHEA treatment stimulated ATPase activity in a dose-dependent manner in young adult rats whereas in the old rats the effect on ATPase activity was marginal. Dehydrogenases activities were somewhat lower in the old rats. DHEA treatment stimulated mitochondrial dehydrogenases activities in both the groups. Results of our studies suggest that judicious use of DHEA treatment can improve oxidative energy metabolism parameters in brain mitochondria from young adult as well as old rats.     

Age-dependent modifications of mitochondrial trans-membrane potential and mass in rat splenic lymphocytes during proliferation

The specific fluorescent probes, Rhodamine 123 (Rh-123) and Nonyl-Acridine Orange (NAO) were, respectively, used to monitor the changes in membrane potential and mass of lymphocyte mitochondria during aging and proliferation. An age-dependent increase of the uptake of both fluorochromes was observed in resting cells; however, NAO fluorescence increased to a greater extent when compared with the Rh-123 probe. This resulted in a lower respiratory activity per unit of mitochondrial mass in old cells than in the young ones. Following mitogenic stimulation, most of the lymphocytes from young rats showed an increase in their membrane potential and mass. On the contrary about 50% of cells from old rats had depolarized mitochondria after 72 h from the stimulation. Present data support that mitochondria of lymphocytes from old rats are extremely sensitive to the stressing conditions resulting from mitogenic stimulation.     

Mechanisms of respiration and phosphorylation in Ascaris muscle mitochondria

In Ascaris muscle mitochondria the major respiratory chain-linked phosphorylation activity is accomplished by a NADH-linked reduction of fumarate to succinate. Oxygen can also be employed as a terminal electron acceptor via a cyanide- and salicyl-hydroxamate-resistant terminal oxidase. As in fumarate-dependent electron transport this process appears to be coupled to energy conservation at phosphorylation site I. The branchpoint from which electrons are taken from the main respiratory chain to either the alternative oxidase or fumarate reductase is likely to be on the oxygen side of the NADH dehydrogenase segment.Malate and succinate are the only substrates which appreciably support respiration in the mitochondrion of the nematode. Regardless of the presence or absence of oxygen malate is utilized by an oxidation-reduction reaction resulting in the formation of pyruvate, acetate, succinate, propionate and CO₂. In addition, aerobically, hydrogen peroxide is formed as the product of oxygen reduction. Succinate accumulation was found to be significantly higher in the anaerobic as compared to the aerobic incubation mixtures. This effect was accompanied by an increase in anaerobic malate consumption. ATP generation and the formation of pyruvate, acetate and propionate were found to be similar in the presence and absence of oxygen.In malate-supported respiration of intact Ascaris mitochondria reducing equivalents (NADH) are produced exclusively through pyruvate and acetate formation. These enzymatic reactions are functionally coupled to the electron transport-linked reductions of fumarate to succinate and oxygen to hydrogen peroxide, respectively. In accordance with the position of the redox potentials of the fumarate/succinate and O₂/H₂O₂ couples, anaerobic and aerobic respiration was found to be associated with relatively low energy conservation efficiencies. Thus one molecule of ATP was conserved per 2e^? transferred to fumarate or oxygen, respectively. No evidence could be obtained for a significant activity of energy conservation sites II and III and electron transfer through the alternative oxidase pathway was shown not to be coupled to phosphorylation.     

Nephrotoxicity of ferric nitrilotriacetate. An electron-microscopic and metabolic study.

Repeated intraperitoneal injections of ferric nitrilotriacetate (Fe-NTA) induce nephrotoxic features such as proximal tubular necrosis and renal failure, an unexpected phenomenon for a ferric compound. The mechanism of Fe-NTA toxicity was investigated by electron microscopy and respiration studies of renal cortical mitochondria in rats. Four hours after a single intraperitoneal injection of Fe-NTA, 5 mg iron/kg body wt, loss of microvilli, increased number of cytoplasmic vacuoles, electron-dense cytoplasmic deposits, mitochondrial swelling, karyorrhexis, and rupture of cytoplasmic membrane were observed in proximal tubular epithelia. At 24 hours, an increased number of cells had become necrotic. Polarographic studies of mitochondria from renal cortex 4 hours after Fe-NTA treatment showed a significant decrease in State 3 respiration and DNP-uncoupled respiration, whereas little change was observed in State 4 respiration and ADP/O.     

Effect of aging and acetyl-L-carnitine on energetic and cholinergic metabolism in rat brain regions

The effect of aging and subchronic treatment with acetyl-L-carnitine (50 mg/kg per day) was studied on mitochondrial bioenergetics and cholinergic metabolism in non-synaptic mitochondria and synaptosomes isolated from cerebral cortex, hippocampus and striatum of rats aged 4, 11 and 18 months. Respiratory activity and cytochrome oxidase specific activity were unaffected by aging in non-synaptic mitochondria. In synaptosomes, pyruvate dehydrogenase, choline acetyltransferase and acetylcholinesterase specific activity remained unchanged, but the high-affinity choline uptake decreased in cerebral cortex and striatum of 18-month-old rats. Acetyl-L-carnitine treatment increased the high-affinity choline uptake in cerebral cortex of 18-month-old rats. The treatment caused also an increase in cytochrome oxidase activity in all the three cerebral regions and in choline uptake in the hippocampus, parameters that were not directly affected by aging processes.     

The relationship between the cardiac contractile function,adenine nucleotides and amino acids of cardiac tissue and mitochondria at acute respiratory hypoxia

The effect of asphyxia and subsequent resumption of respiration on the content of adenine nucleotides and some amino acids in heart tissue and mitochondria, as well as respiration of heart mitochondria was studied in rats. The depression of cardiac contractile function during asphyxia showed a better correlation with losses in mitochondrial adenine nucleotides (ATP+ADP+AMP) than those in cardiac tissue. The decrease in the heart work index was accompanied by a decrease in state 3 respiration with glutamate and malate as well as uncoupled respiration with these substrates. This did not occur with succinate. Nonphosphorylating (state 4) respiratory rates and ADP/O ratios were slightly affected by asphyxia, when respiratory substrates of both types were used. The decreased level of glutamic acid in the tissue and mitochondria of asphyxic hearts was simultaneously observed with a significant increase of alanine in cardiac tissue and of aspartic acid in the mitochondria. The losses of intramitochondrial ATP and respiratory activity with NAD-dependent substrates during asphyxia were associated with a reduction of glutamic acid level in mitochondria. The recovery of cardiac function during resumption of respiration was related to the restoration of mitochondrial respiration supported by glutamate and malate, as well as to the restoration of mitochondrial adenine nucleotides and glutamic acid. The results suggest that the depression of cardiac function caused by acute respiratory hypoxia may be attributed to impairment of electron transport, particularly in complex I of the respiratory chain and changes in metabolism of glutamic acid.     

Age-related changes in mitochondrial respiration and oxidative damage in the cerebral cortex of the Fischer 344 rat

This study probed possible age-related changes in mitochondrial bioenergetics in naïve Fischer 344 rats. Synaptic and extrasynaptic mitochondria were isolated from the cortex of one hemisphere of young (3-5 months), middle (12-14 months), or aged (22-24 months) rats. Respiration parameters were obtained using a Clarke-type electrode. Aged rats displayed no significant alterations in respiration, indicating mitochondria must be more resilient to the aging process than previously thought. Synaptic mitochondria displayed lower respiration capacities than the extrasynaptic fraction. Aged F344 rats appear capable of normal electron transport chain function without declines in ability to produce ATP. Markers of cortical oxidative damage (3-nitrotyrosine [3-NT], 4-hydroxynonenal [4-HNE], and protein carbonyls [PC]) were collected from the post-mitochondrial supernatant (PMS) from the contralateral hemisphere, and from mitochondrial samples following respiration analysis. Age-related increases in PC and 3-NT levels were found in synaptic mitochondria, whereas significant extrasynaptic elevations were only found in middle aged rats. These findings support an age-related increase in oxidative damage in the cortex, while proposing the two fractions of mitochondria are differentially affected by the aging process. Levels of oxidative damage that accumulates in the cortex with age does not appear to significantly impair cortical mitochondrial respiration of F344 rats.     

Anomalous expression of microtubule-associated protein 1B in the hippocampus and cortex of aged rats treated with pentylenetetrazole

The aim of the present study was to assess the age-dependent response of microtubule-associated protein 1B, a plasticity-associated protein deriving from a late gene, following administration of an epileptogenic stimulus. The effect of a single administration of the convulsant pentylenetetrazole on microtubule-associated protein 1B expression in the hippocampal formation and cortex of three-, 18- and 28-month-old rats was assessed using northern blot analysis, in situ hybridization and immunohistochemistry. In three-month-old rats, we detected initial increases in microtubule-associated protein 1B messenger RNA at 15 h following pentylenetetrazole administration in the granule cells of the dentate gyrus, in the CA3 region of the hippocampus and in layers II/III of the entorhinal cortex, and these reached a maximum at 44 h. However, in the hippocampus and cortex of 18-month-old rats, the peak occurred at 15 h, and in the brains of 28-month-old rats a blunted peak was reached at 3 h. Pentylenetetrazole treatment in young rats resulted in a robust induction of microtubule-associated protein 1B immunoreactivity in the granule cells of the dentate gyrus and in layers II/III of the entorhinal cortex, but also produced a large decrease in the retrosplenial cortex. However, following pentylenetetrazole treatment in older rats, the granule cells of the dentate gyrus were nearly devoid of microtubule-associated protein 1B immunoreactivity, whereas the retrosplenial cortex showed no changes at all, and the entorhinal cortex had an expression pattern similar to that of young rats. Aberrant immunolabeling of microtubule-associated protein 1B occurred in cortical layer VI of the aged rats where, unlike in young rats, there was heavy staining of neuronal somata. These results suggest that the regulation of the plasticity-associated protein microtubule-associated protein 1B is altered in the ageing rat brain, with the peak of expression shifted to earlier times in 18-month-old rats and blunted, variable increases at even earlier times in 28-month-old rats.