Christine Peterson

Calcium and the aging nervous system

Many aspects of calcium homeostasis change with aging. Numerous calcium compartments complicate studies of altered calcium regulation. However, age-related decreases in calcium permeation across membranes and mobilization from organelles may be a common fundamental change. Deficits in ion movements appear to lead to altered coupling of calcium-dependent biochemical and neurophysiological processes and may lead to pathological and behavioral changes. The calcium-associated changes during aging probably do not occur with equal intensity in all cell types or in different parts of the same cell. Thus, cells or compartments with a high proportion of calcium activated processes would be more sensitive to diminished calcium availability. These age-related changes may predispose the brain to the development of age-related neurological disorders. The effects of decreased ion movement may be further aggravated by an age-related decline in other calcium-dependent processes. Depression of some of these calcium-dependent functions appears physiologically significant, since increasing calcium availability ameliorates age-related deficits in neurotransmission and behavior. A better understanding of the interactions between calcium homeostasis and calcium-dependent processes during aging will likely help in the design of more effective therapeutic strategies.

Aging Decreases Oxidative Metabolism and the Release and Synthesis of Acetylcholine

Acetylcholine (ACh) synthesis in vivo is known to decrease during the aging process (senescence). To elucidate the molecular mechanism(s) of this age‐related decline, we studied brain slices from 3‐, 10‐, and 30‐month‐old mice of two strains (C57B1 and Balb/c). In low K+ media, oxidative metabolism as measured by 14CO2 production decreased with aging from 100% (3 months) to 85% (10 months) or 71% (30 months) whether [U−14C]glucose, [3,4‐14C]glucose, or [l‐14C]pyruvate was the substrate. In the aged brain (3 months) the increase in 14CO2 production with K+ stimulation was about twofold higher than in the young brain (3 months). Thus, in high K+ media, only slight decreases (<10%) in oxidative metabolism occurred with aging. Changes in ACh synthesis paralleled the decreases in 14CO2 production. Synthesis of [14C]ACh from [U‐14C]glucose in low K+ media declined from 100% (3 months) to 85% (10 months) or 66% (30 months), while in high K+ media only slight decreases (<10.5%) occurred with aging. The Ca2+‐dependent, K+‐stimulated release of [14C]ACh declined from 100% (3 months) to 58% (10 months) or 25% (30 months). Only the decrease in the release of ACh declined to the same extent as the reduced in vivo synthesis of ACh with aging. The results suggest that decreases in oxidative metabolism, ACh synthesis, and in the release of ACh contribute to a reduction in cholinergic function in the senescent brain.

Decreases in Amino Acid and Acetylcholine Metabolism During Hypoxia

Abstract: Hypoxia impairs brain function by incompletely defined mechanisms. Mild hypoxia, which impairs memory and judgment, decreases acetylcholine (ACh) synthesis, but not the levels of ATP or the adenylate energy charge. However, the effects of mild hypoxia on the synthesis of the glucosederived amino acids [alanine, aspartate, γ‐amino butyric acid (GABA), glutamate, glutamine, and serine] have not been characterized. Thus, we examined the incorporation of [U‐14C]glucose into these amino acids and ACh during anemic hypoxia (injection of NaNO2), hypoxic hypoxia (15 or 10% O2), and hypoxic hypoxia plus hypercarbia (15 or 10% O2 with 5% CO2). In general, the synthesis of the amino acids and of ACh declined in parallel with each type of hypoxia we studied. For example, anemic hypoxia (75 mg/kg of NaNO2) decreased the incorporation of [U‐14C]glucose into the amino acids and into ACh similarly. [Percent inhibition: ACh (57.4), alanine (34.4), aspartate (49.2), GABA (61.9). glutamine (59.2), glutamate (51.0), and serine (36.7)]. A comparison of several levels (37.5, 75, 150, 225 mg/kg of NaNO2) of anemic hypoxia showed a parallel decrease in the flux of glucose into ACh and into the amino acids whose synthesis depends on mitochondrial oxidation: GABA (r= 0.98), glutamate (r= 0.99), aspartate (r= 0.96), and glutamine (r= 0.97). The synthesis of the amino acids not dependent on mitochondrial oxidation did not correlate as well with changes in ACh metabolism: serine (r= 0.68) and alanine (r= 0.76). The decreases in glucose incorporation into ACh and into the amino acids with hypoxic hypoxia (15% or 10% O2) or hypoxic hypoxia with 5% CO2 were very similar to those with the two lowest levels of anemic hypoxia. Thus, any explanation of the brains sensitivity to a decrease in oxygen availability must include the alterations in the metabolism of the amino acid neurotransmitters as well as ACh.

Neurotransmitter and carbohydrate metabolism during aging and mild hypoxia

Alterations in the metabolism of the glucose derived neurotransmitters may underlie some of the deficits in brain function that can accompany aging. We examined the whole brain syntheses of acetylcholine (ACh), alanine, aspartate, glutamate, gamma-aminobutyrate (GABA), glutamine and serine in two strains (C57BL and BALB/c) of aged mice (3, 10 and 30 months). ACh synthesis in C57BL and BALB/c mice declined 41 and 44% at 10 months and 64 and 75% by 30 months. Incorporation of [U-14C]glucose into amino acids generally decreased with aging, but it was not depressed as much as ACh formation. The only significant reductions in the amino acids in the 30 month old mice of both strains were in the syntheses of GABA (46 and 32%) and glutamine (44 and 55%). These changes may make the aged brain more vulnerable to metabolic insults, since mild anemic hypoxia decreased the syntheses of all the neurotransmitters at all ages even further. ACh synthesis in hypoxic 30 month old mice was only 9-11% of the 3 month old nonhypoxic mice, whereas amino acid formation ranged from 18-55% of the 3 month old nonhypoxic mice. Carbohydrate metabolism and its response to metabolic insults was also altered by age in both strains. The 30 month old mice had higher brain lactate concentrations than the 3 month old mice. The combination of hypoxia and aging further depressed oxidative metabolism, since a greater increase in brain lactates occurred in the aged hypoxilism and its response to metabolic insults was also altered by age in both strains. The 30 month old mice had higher brain lactate concentrations than the 3 month old mice. The combination of hypoxia and aging further depressed oxidative metabolism, since a greater increase in brain lactates occurred in the aged hypoxilism and its response to metabolic insults was also altered by age in both strains. The 30 month old mice had higher brain lactate concentrations than the 3 month old mice. The combination of hypoxia and aging further depressed oxidative metabolism, since a greater increase in brain lactates occurred in the aged hypoxic mice than in young hypoxic mice. Thus, aging may reduce the ability of the brain to adapt to metabolic insults.

Amelioration of age-related neurochemical and behavioral deficits by 3,4-diaminopyridine

Alterations of the cholinergic system may be responsible for age-related changes in behavior. The in vitro calcium dependent release of acetylcholine and tight rope test performance decline in parallel during senescence. 3,4-Diaminopyridine, which enhances calcium influx by nerve terminals, diminishes these age-related alterations. In aged mice (30 month old), 3,4-diaminopyridine increases the calcium dependent release of acetylcholine (260%) and improves tight rope test performance (428%). These results support the hypothesis that alterations in calcium homeostasis underlie some of the cholinergic and behavioral deficits that accompany senescence.

Subsynaptosomal distribution of calcium during aging and 3,4-diaminopyridine treatment

Since previous studies showed that calcium uptake by synaptosomes from rodents declines with aging, the subsynaptosomal distribution of calcium was determined with the disruption method of Scott et al. Calcium uptake by the mitochondrial (digitonin-resistant) and non-mitochondrial (digitonin-labile) compartments, as well as total uptake, were determined at 2, 5 and 10 min. After a 10 min incubation under resting conditions (5 mM-KCl), total calcium uptake decreased at 10 months (-14.6%) and 30 months (-33.0%) of age; mitochondrial calcium uptake increased by 10 months (+ 11.2%) but declined by 30 months (-17.5%); the non-mitochondrial calcium compartment declined at 10 (-34.7%) and 30 (-43.4%) months when compared to the 3 month old control. With potassium depolarization (31 mM-KCl), total calcium uptake declined from 100% (3 months) to 73.8% (10 months) or 53.0% (30 months); mitochondrial calcium uptake declined from 100% (3 months) to 85.6% (10 months) or 68.4% (30 months); non-mitochondrial calcium uptake decreased at 10 (-34.3%) and 30 (-57.7%) months of age when compared to 3 months (100%). The deficits in calcium homeostasis are not due to changes in synaptosomal volumes or to diminished membrane potentials, as assessed by tetraphenylphosphonium ion accumulation. 3,4-Diaminopyridine partially reversed the alterations in total, mitochondrial and non-mitochondrial calcium uptake by synaptosomes from aged mice.

Synaptosomal Calcium Metabolism During Hypoxia and 3,4‐Diaminopyridine Treatment

Abstract: A decline in the calcium‐dependent release of neurotransmitters appears to underlie the decreased neuronal function that accompanies reduced oxygen tensions (hypoxia). To determine if alterations in calcium uptake are primary to these changes, synaptosomal calcium uptake was measured in the presence of 100%, 2.5%, or 0% oxygen. Calcium uptake declined 60.2 ± 0.1 and 82.4 ± 2.5% with 2.5% and 0% when compared with 100% oxygen, respectively. 3,4‐Diaminopyridine stimulated calcium uptake by synaptosomes when they were incubated in low‐potassium media. It also diminished the hypoxic‐induced decline in calcium uptake to 30.6 ± 3.1 and 33.5 ± 3.1% with 2.5% and 0% oxygen, respectively. External binding to the synaptosomal plasma membrane declined to 29.2 ± 0.3 or 11.8 ± 0.9% when the oxygen tension was reduced to 2.5% or 0% oxygen. 3,4‐Diaminopyridine increased this superficial binding from 111.7 ± 0.3 to 86.5 ± 0.9 or 23.4 ± 0.9% with 100%, 2.5%, or 0% oxygen when compared with 100% oxygen without 3,4‐diaminopyridine, respectively. Thus, the decline in neuronal processing that accompanies acute hypoxia may be due to altered calcium homeostasis, which diminishes neuro‐transmitter release.

Cholinergic drugs and 4-aminopyridine alter hypoxic-induced behavioral deficits

To test the hypothesis that decreased acetylcholine (ACh) metabolism during hypoxia is behaviorally important, the effects of cholinergic drugs and 4-aminopyridine, an enhancer of ACh release, were examined in hypoxic mice. Chemical hypoxia (150 mg/kg NaNO2) reduced tight rope test scores from 13.2 +/- 0.2 to 2.8 +/- 0.3. 4-Aminopyridine partially reversed the scores of hypoxic mice (7.3 +/- 0.7). Physostigmine improved performance by hypoxic mice (7.9 +/- 0.4) when it was given before NaNO2 and this effect was blocked by pretreatment with atropine (3.5 +/- 0.4) or mecamylamine (2.4 +/- 0.6). Neostigmine (0.002-0.2 mg/kg) was ineffective. Performance also improved if atropine (7.0 +/- 0.5) or mecamylamine (7.2 +/- 0.5) was given before NaNO2. Nicotine (5.5 +/- 0.7) or the muscarinic agonist arecoline (5.4 +/- 0.5) improved performance when given during the hypoxic episode, and when the drugs were combined, the score was even higher (8.2 +/- 1.0). Neither epinephrine (0.002-2.0 mg/kg) nor norepinephrine (0.00002-2.0 mg/kg) improved performance by hypoxic mice. These results suggest that hypoxia produces a behaviorally important impairment of the cholinergic system perhaps through a primary alteration of acetylcholine release.

Subsynaptosomal Calcium Distribution During Hypoxia and 3,4-Diaminopyridine Treatment

Abstract: Previous results demonstrate that hypoxia (low oxygen) diminishes calcium uptake by synaptosomes. The present studies examined the effects of low oxygen on calcium homeostasis in the digitonin‐resistant (mitochondrial) and the digitonin‐labile (nonmitochondrial) compartments of intact synaptosomes and their relation to altered membrane potentials. A 10‐min hypoxic incubation in low‐potassium media reduced total (‐38.3%), mitochondrial (‐43.3%), and nonmitochondrial (‐27.8%) calcium uptake. In high‐potassium media, low oxygen reduced mitochondrial (‐41.2%) and total (‐34.4%) uptake whereas nonmitochondrial (+6%) calcium uptake was essentially unaffected. A temporal analysis of nonmitochondrial calcium uptake revealed an initial depression (0–5 min) followed by a stimulation (5–10 min). Hypoxic‐induced alterations in the subsynaptosomal distribution of calcium resembled those produced by uncouplers [FCCP (carbonylcyanide‐p‐trifluoro‐methoxyphenylhydrazone) or rotenone plus oligomycin]. 3,4‐Diaminopyridine partially ameliorated the hypoxic‐and FCCP‐induced decreases in synaptosomal calcium uptake. Low oxygen reduced the total synaptosomal membrane potential (i.e., plasma plus mitochondrial membrane potential) as measured by an increased efflux of tetraphenylphosphonium ion. This hypoxic‐induced efflux of tetraphenylphosphonium was slowed by pretreatment with 3,4‐diaminopyridine. Thus, both drug and membrane potential studies suggest that hypoxic‐induced alterations in the subcellular distribution of calcium may be due to an uncoupling mechanism and a collapse of the synaptosomal mitochondrial membrane potential.

Excitatory Amino Acids and Hepatic Encephalopathy

There is substantial evidence to suggest that glutamate and aspartate are excitatory neurotransmitters in the mammalian central nervous system. Glutamate content of brain is decreased in experimental models of acute and chronic hyperammonemia as well as in autopsied brain tissue from patients dying in hepatic coma resulting from fulminant hepatic failure or cirrhosis. Glutamate-related enzyme changes have also been observed in brain in both experimental and human hepatic encephalopathy. Exposure of hippocampal slices to pathophysiological concentrations of ammonia did not result in significant decreases of K+-stimulated Ca2+-dependent release of glutamate suggesting that the synthesis of neurotransmitter amino acid was not affected in hyperammonemia. However, evoked release of glutamate from hippocampal slices from 4-week portacaval shunted rats was increased 2-fold. Assessment of glutamate receptors by quantitative autoradiography revealed widespread reductions of the N-Methyl-D-Aspartate (NMDA) subclass of receptors. Taken together with the increased glutamate release of in this material, these results suggest that extracellular glutamate (or aspartate) may be increased in sustained hyperammonemia, probably as a result of loss of astrocytic integrity. Such changes could be of pathophysiological significance in hepatic encephalopathy.