Intracellular pH buffering shapes activity-dependent Ca2+ dynamics in dendrites of CA1 interneurons.

Voltage-gated calcium (Ca) channels are highly sensitive to cytosolic H+, and Ca2+ influx through these channels triggers an activity-dependent fall in intracellular pH (pHi). In principle, this acidosis could act as a negative feedback signal that restricts excessive Ca2+ influx. To examine this possibility, whole cell current-clamp recordings were taken from rat hippocampal interneurons, and dendritic Ca2+ transients were monitored fluorometrically during spike trains evoked by brief depolarizing pulses. In cells dialyzed with elevated internal pH buffering (high beta), trains of >15 action potentials (Aps) provoked a significantly larger Ca2+ transient. Voltage-clamp analysis of whole cell Ca currents revealed that differences in cytosolic pH buffering per se did not alter baseline Ca channel function, although deliberate internal acidification by 0.3 pH units blunted Ca currents by approximately 20%. APs always broadened during a spike train, yet this broadening was significantly greater in high beta cells during rapid but not slow firing rates. This effect of internal beta on spike repolarization could be blocked by cadmium. High beta also 1) enhanced the slow afterhyperpolarization (sAHP) seen after a spike train and 2) accelerated the decay of an early component of the sAHP that closely matched a sAHP conductance that could be blocked by apamin. Both of these effects on the sAHP could be detected at high but not low firing rates. These data suggest that activity-dependent pHi shifts can blunt voltage-gated Ca2+ influx and retard submembrane Ca2+ clearance, suggesting a novel feedback mechanism by which Ca2+ signals are shaped and coupled to the level of cell activity.

Similar propagation of SD and hypoxic SD-like depolarization in rat hippocampus recorded optically and electrically.

Neuron membrane changes and ion redistribution during normoxic spreading depression (SD) induced, for example, by potassium injection, closely resemble those that occur during hypoxic SD-like depolarization (HSD) induced by oxygen withdrawal, but the degree to which the two phenomena are related is controversial. We used extracellular electrical recording and imaging of intrinsic optical signals in hippocampal tissue slices to compare 1) initiation and spread of these two phenomena and 2) the effects of putative gap junction blocking agents, heptanol and octanol. Both events arose focally, after which a clear advancing wave front of increased reflectance and DC shift spread along the CA1 stratum radiatum and s. oriens. The rate of spread was similar: conduction velocity of normoxic SD was 8.73 +/- 0.92 mm/min (mean +/- SE) measured electrically and 5.84 +/- 0.63 mm/min measured optically, whereas HSD showed values of 7.22 +/- 1.60 mm/min (electrical) and 6.79 +/- 0.42 mm/min (optical). When initiated in CA1, normoxic SD consistently failed to enter the CA3 region (7/7 slices) and could not be initiated by direct KC1 injection in the CA3 region (n = 3). Likewise, the hypoxic SD-like optical signal showed onset in the CA1 region and halted at the CA1/CA3 boundary (9/9 slices), but in some (4/9) slices the dentate gyrus region showed a separate onset of signal changes. Microinjection into CA1 stratum radiatum of octanol (1 mM), which when bath applied arrests the spread of normoxic SD, created a small focus that appeared to be protected from hypoxic depolarization. However, bath application of heptanol (3 mM) or octanol (2 mM) did not prevent the spread of HSD, although the onset was delayed. This suggests that, although gap junctions may be essential for the spread of normoxic SD, they may play a less important role in the spread of HSD.

Differential sensitivity to intracellular pH among high- and low-threshold Ca2+ currents in isolated rat CA1 neurons.

The effects of intracellular pH (pHi) on high-threshold (HVA) and low-threshold (LVA) calcium currents were examined in acutely dissociated rat hippocampal Ca1 neurons with the use of the whole cell patch-clamp technique (21-23 degrees C). Internal pH was manipulated by external exposure to the weak base NH4Cl or in some cases to the weak acid Na-acetate (20 mM) at constant extracellular pH (7.4). Confocal fluorescence measurements using the pH-sensitive dye SNARF-1 in both dialyzed and intact cells confirmed that NH4Cl caused a reversible alkaline shift. However, the external TEA-Cl concentration used during ICa recording was sufficient to abolish cellular acidification upon NH4Cl wash out. With 10 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) in the pipette, NH4Cl exposure reversibly enhanced HVA currents by 29%, whereas exposure to Na-acetate markedly and reversibly depressed HVA Ca currents by 62%. The degree to which NH4Cl enhanced HVA currents was inversely related to the internal HEPES concentration but was unaffected when internal ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) was replaced by equimolar bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid (BAPTA). When depolarizing test pulses were applied shortly after break-in (Vh = -100 mV), NH4Cl caused a proportionally greater increase in the sustained current relative to the peak. The dihydropyridine Ca channel antagonist nifedipine (5 microM) blocked nearly all of this sustained current. A slowly inactivating nifedipine-sensitive (L-type) HVA current could be evoked from a depolarized holding potential of -50 mV; NH4Cl enhanced this current by 40 +/- 3% (mean +/- SE) and reversibly shifted the tail-current activation curve by +6-8 mV. L-type currents exhibited more rapid rundown than N-type currents; HVA currents remaining after prolonged cell dialysis, or in the presence of nifedipine, inactivated rapidly and were depressed by omega-conotoxin (GVIA). NH4Cl enhanced these N-type currents by 76 +/- 9%. LVA Ca currents were observed in 32% of the cells and exhibited little if any rundown. These amiloride-sensitive currents activated at voltages negative to -50 mV, were enhanced by extracellular alkalosis and depressed by extracellular acidosis, but were unaffected by exposure to either NH4Cl or NaAC. These results demonstrate that HVA Ca currents in hippocampal CA1 neurons are bidirectionally modulated by internal pH shifts, and that N-type currents are more sensitive to alkaline shifts than are L- or T-type (N > L > T). Our findings strengthen the idea that distinct cellular processes governed by different Ca channels may be subject to selective modulation by uniform shifts in cytosolic pH.

Heptanol but not fluoroacetate prevents the propagation of spreading depression in rat hippocampal slices.

We investigated whether heptanol and other long-chain alcohols that are known to block gap junctions interfere with the generation or the propagation of spreading depression (SD). Waves of SD were triggered by micro-injection of concentrated KCl solution in stratum (s.) radiatum of CA1 of rat hippocampal tissue slices. DC-coupled recordings of extracellular potential (V0) were made at the injection and at a second site approximately 1 mm distant in st. radiatum and sometimes also in st. pyramidale. Extracellular excitatory postsynaptic potentials (fEPSPs) were evoked by stimulation of the Schaffer collateral bundle; in some experiments, antidromic population spikes were evoked by stimulation of the alveus. Bath application of 3 mM heptanol or 5 mM hexanol completely and reversibly prevented the propagation of the SD-related potential shift (delta V0) without abolishing the delta V0 at the injection site. Octanol (1 mM) had a similar but less reliably reversible effect. fEPSPs were depressed by approximately 30% by heptanol and octanol, 65% by hexanol. Antidromic population spikes were depressed by 30%. In isolated, patchclamped CA1 pyramidal neurons, heptanol partially and reversibly depressed voltage-dependent Na currents possibly explaining the slight depression of antidromic spikes and, by acting on presynaptic action potentials, also the depression of fEPSPs. Fluoroacetate (FAc), a putative selective blocker of glial metabolism, first induced multiple spike firing in response to single afferent volleys and then severely suppressed synaptic transmission (confirming earlier reports) without depressing the antidromic population spike. FAc did not inhibit SD propagation. The effect of alkyl alcohols is compatible with the idea that the opening of normally closed neuronal gap junctions is required for SD propagation. Alternative possible explanations include interference with the lipid phase of neuron membranes. The absence of SD inhibition by FAc confirms that synaptic transmission is not necessary for the propagation of SD, and it suggests that normally functioning glial cells are not essential for SD generation or propagation.

Salutary and deleterious effects of acidity on an indirect measure of metabolic rate and ATP concentrations in CNS cultures.

Acidosis has traditionally been considered to mediate certain types of hypoxic-ischemic injury to the brain. However, the recent demonstration that moderate acidosis will reduce NMDA-mediated currents suggested that acidity could actually protect against types of ischemia and excitotoxicity, and in vitro studies now support this idea. Prompted by this, we have utilized the silicon microphysiometer, a recently-developed instrument that allows for indirect real-time measurement of metabolic rate by detecting proton efflux from small numbers of cultured cells, to determine whether acidity has protective effects upon cellular metabolism. Reducing extracellular pH from 7.4 to as low as 6.0 caused prompt, step-wise, and reversible inhibition of proton efflux rate in cortical and hippocampal cultures both normally and restricted to either glycolysis or oxidative metabolism. Approximately half of the inhibition was due to acidotic effects of NMDA-mediated currents, as demonstrated with NMDA receptor antagonists. Such an inhibition of this indirect metabolic measure could be associated with constant or increased ATP concentrations and represent a beneficial decrease in energy demands upon a neuron. Alternatively, an inhibition of proton efflux rate could be associated with ATP depletion and reflect impaired energy production. We observed a complex interplay between these opposing patterns. Reducing pH to 6.7 for 20 min caused significantly increased ATP concentrations, and prevented excitotoxin-induced ATP depletion. These effects of acidosis involved both NMDA-dependent and- independent actions. More severe (less than pH 6.7) acidosis did not cause ATP concentrations to rise, and if sustained for more than an hour caused a significant decline in ATP concentrations. Thus, despite the recent emphasis on the surprising neuroprotective potential of acidosis, a drop in pH is still likely to have complex and mixed consequences for brain tissue.

Effects of extracellular pH on voltage-gated Na+, K+ and Ca2+ currents in isolated rat CA1 neurons.

1. The effects of extracellular H+ (pHo) in the pathophysiological range (pH 6-8) on voltage-gated sodium, potassium, and calcium currents were examined in acutely dissociated rat hippocampal CA1 neurons using the whole-cell patch clamp technique. All experiments were conducted in Hepes-buffered solutions and were performed at room temperature (21-23 degrees C). 2. TTX-sensitive sodium currents, evoked by both step and ramp depolarization, were reversibly depressed by moderate acidosis and enhanced slightly by alkaline exposure. Changes in current amplitude were coincident with small reversible shifts (+/- 3 mV) in the voltage dependence of activation. In contrast, sodium current activation and decay kinetics as well as steady-state inactivation were unaffected by acidosis. 3. Outward potassium currents could be separated into a transient, rapidly inactivating current (IA) and a sustained, slowly inactivating component (IK). Steady-state activation of both currents was unaffected by an increase or decrease in pHo. Similarly, IK activation and IA decay kinetics remained stable during pHo exchange. In contrast, the steady-state inactivation (h infinity) of both potassium currents was reversibly shifted by approximately +10 mV during acid exposure, but remained unchanged during alkaline treatment. 4. Calcium currents were found to be predominantly of the high-voltage-activated (HVA) type, which could be carried by Ba2+ and inhibited completely by cadmium. Moderate acidosis (pH 6.9-6.0) reversibly depressed HVA Ca2+ current amplitude and caused a positive shift in its voltage dependence. For both of these parameters, alkaline treatment (pH 8.0) had the opposite effect. The depression of HVA Ca2+ currents by low pHo was unaffected by raising the internal Hepes concentration from 10 to 50 mM in the patch pipette. A Hill plot of the effect of pH on Ca2+ current amplitude revealed a pK value (defined as the mid-point of the titration curve) of 7.1 and a slope of 0.6. 5. The rate of Ca2+ current activation was unaffected by pHo at positive potentials, but below 0 mV the activation rate increased at low pH and decreased at high pH, becoming significant at -20 mV. At this membrane voltage, a second HVA current was revealed during acid exposure as the whole-cell HVA current was depressed. Ca2+ current decay was described by two time constants, both of which were significantly reduced at pH 6.4 and slightly enhanced at pH 8.0. Steady-state Ca2+ current inactivation reached 50% near -50 mV and was not affected at either pH extreme. 6. These results demonstrate that extracellular pH shifts within the pathophysiological range are capable of modulating both the conductance and gating properties of voltage-gated ion channels in hippocampal CA1 neurons. The effects we describe are consistent with the wellknown effects of pHo on neuronal excitability and strengthen the idea that endogenous pHo shifts may help regulate cell activity in situ.

Mild acidosis delays hypoxic spreading depression and improves neuronal recovery in hippocampal slices.

Severe tissue acidosis has been viewed traditionally as a damaging component of cerebral hypoxia. However, a neuroprotective action of low pH during hypoxia has been described in primary neuronal cultures. To identify and characterize this effect in mature brain tissue, adult rat hippocampal slices were made hypoxic after adjusting pHo with HCl or NaOH. Ion-selective microelectrodes were positioned in CA1 to record evoked field potentials, extracellular DC voltage (Vo), pHo, and [Ca2+]o. Orthodromic population spike amplitude was used as a measure of slice recovery 2 hr after reoxygenation. All slices became markedly acidotic during hypoxia (delta pHo approximately 0.4 pH unit). Following restoration of O2 and bath pH to 7.4, slice pHo returned to its pretreatment level regardless of experimental treatment, hypoxic duration, or the degree of electrophysiological recovery. When either the period of hypoxia or the duration of HSD was held constant, acid-treated slices exhibited a significant improvement in recovery. However, in neither paradigm did the recovery of alkaline-treated slices differ from controls. Mild acidosis (bath pH = 6.9-7.3) caused a reversible depression of the orthodromic population spike, an increase in the latency of hypoxic spreading depression-like depolarization (HSD), and a decrease in the magnitude of the associated negative Vo shift. For each of these parameters, mild alkalinity (bath pH = 7.7) had the opposite effect. Acid treatment did not affect the decrease in [Ca2+]o during HSD but accelerated its recovery after reoxygenation. These results suggest that mild acidosis may limit hypoxic neuronal injury in vitro by delaying HSD onset and by additional mechanisms unrelated to the degree of calcium influx during neuronal depolarization.

Evolving concepts about the role of acidosis in ischemic neuropathology.

Cerebral ischemia is one of the most common neurological insults. Many pathological events are undoubtedly triggered by ischemia, but only recently has it become accepted that ischemic cell injury arises from a complex interaction between multiple biochemical cascades. Tissue acidosis is a well established feature of ischemic brain tissue, but its role in ischemic neuropathology is still not fully understood. Within the last few years, new evidence has challenged the historically negative view of acidosis and suggests that it may play more of a beneficial role than previously thought. This review reintroduces the concept of acidosis to ischemic brain injury and presents some new perspectives on its neuroprotective potential.

Endocrine features of glucocorticoid endangerment in hippocampal astrocytes.

Metabolic insults, such as ischemia or hypoglycemia, typically cause severe neuronal injury in the hippocampus and this cell vulnerability can be exacerbated by glucocorticoid (GC) exposure. This endangerment can also be demonstrated in vitro in both neurons and astrocytes. Direct GC effects on cell physiology thus appear to play a role, but the actual mechanism remains unclear. In order to clarify whether GCs act as damaging agents via a 'classical' steroid route, we examined the temporal features and steroid-specificity of this synergy in hippocampal astrocyte cultures derived from E18 fetal rats. A 24-hour pretreatment with corticosterone (CORT), the principal GC in the rat, enhanced both hypoxic and hypoglycemic cell damage, as measured by lactate dehydrogenase assay. This damaging effect was abolished when CORT exposure was reduced to 8 or 4 h prior to the hypoglycemic or hypoxic treatment, respectively. A 24-hour exposure to several nonGC steroids also failed to enhance hypoxic cell damage. The damaging effect of CORT was attenuated if steroid exposure occurred during the hypoglycemic insult and was absent in both hypoxic and hypoglycemic paradigms if CORT exposure was limited to the recovery period. These results suggest that GCs aggravate metabolic astrocyte injury via classical hormonal effects that are steroid-specific, receptor-mediated, and emerge slowly after prolonged steroid exposure.

Corticosterone accelerates hypoxia- and cyanide-induced ATP loss in cultured hippocampal astrocytes.

Glucocorticoids potentiate injury to the rodent hippocampus following a variety of metabolic insults, including hypoxia/ischemia, both in vitro and in vivo. We have examined whether corticosterone (CORT), the principal glucocorticoid in the rat, could exacerbate hypoxic energy failure in cultured hippocampal astrocytes. Exposure to 6 h of atmospheric hypoxia (100% N2) or to 30 min of cyanide did not cause any detectable cell injury, although moderate astrocyte damage did occur alter 6 h of hypoxia in the absence of glucose. Both cyanide and hypoxia significantly reduced astrocyte ATP content, a decline that was further reduced when glucose was omitted. A 30 min exposure to 100 microM glutamate elevated ATP content under normoxic conditions but enhanced the cyanide-induced loss of ATP. A 24 h pre-treatment with CORT did not influence normoxic ATP levels but potentiated the loss of ATP following both cyanide and hypoxia. CORT also exacerbated the loss of ATP seen after combined exposure to cyanide and glutamate, as well as that following cyanide + 0 mM glucose. These results indicate that both CORT and glutamate can potentiate hypoxia-induced energy failure in hippocampal astrocytes, albeit by different mechanisms.

Glucocorticoids exacerbate hypoxic and hypoglycemic hippocampal injury in vitro: biochemical correlates and a role for astrocytes.

The acute secretion of glucocorticoids is critical for responding to physiological stress. Under normal circumstances these hormones do not cause acute neuronal injury, but they have been shown to enhance ischemic and seizure-induced neuronal injury in the rat brain. Using fetal rat hippocampal cultures, we asked whether hypoxic and hypoglycemic cell damage in vitro could be exacerbated by direct exposure to corticosterone (CORT). Each of these insults alone damaged neuronal cells, whereas 4-6 h of hypoxic treatment could damage age-matched astrocytes if glucose was reduced or omitted. Ischemic-like injury to both cell types could be attenuated by pretreatment with high (30 mM) glucose. Exposure to 100 nM CORT did not affect cell viability under control conditions but enhanced both hypoxic and hypoglycemic neuronal injury. In both cases, pretreatment with high glucose abolished this CORT-mediated synergy. In astrocyte cultures, CORT exacerbated both hypoxic and hypoglycemic injury and this effect was also attenuated by high-glucose pretreatment. Identical 24-h CORT treatment caused a 13% reduction in glucose uptake in astrocytes and a 38% reduction in glycogen content, without affecting the level of intracellular glucose. Thus, CORT could endanger both neurons and astrocytes in mixed hippocampal cultures and this effect emerged only under conditions of substrate depletion. The metabolic disruption in astrocytes by CORT further suggests that the ability of CORT to exacerbate neuronal injury may be due in part to impaired glial cell function.

Glucocorticoids inhibit glucose transport and glutamate uptake in hippocampal astrocytes: implications for glucocorticoid neurotoxicity.

Glucocorticoids (GCs), the adrenal steroid hormones secreted during stress, can damage the hippocampus and impair its capacity to survive coincident neurological insults. This GC endangerment of the hippocampus is energetic in nature, as it can be prevented when neurons are supplemented with additional energy substrates. This energetic endangerment might arise from the ability of GCs to inhibit glucose transport into both hippocampal neurons and astrocytes. The present study explores the GC inhibition in astrocytes. (1) GCs inhibited glucose transport approximately 15-30% in both primary and secondary hippocampal astrocyte cultures. (2) The parameters of inhibition agreed with the mechanisms of GC inhibition of glucose transport in peripheral tissues: A minimum of 4 h of GC exposure were required, and the effect was steroid specific (i.e., it was not triggered by estrogen, progesterone, or testosterone) and tissue specific (i.e., it was not triggered by GCs in cerebellar or cortical cultures). (3) Similar GC treatment caused a decrease in astrocyte survival during hypoglycemia and a decrease in the affinity of glutamate uptake. This latter observation suggests that GCs might impair the ability of astrocytes to aid neurons during times of neurologic crisis (i.e., by impairing their ability to remove damaging glutamate from the synapse).

Mechanistic distinctions between excitotoxic and acidotic hippocampal damage in an in vitro model of ischemia.

Excitotoxicity is believed to underlie the selective loss of vulnerable neurons after transient ischemia, while lactic acidosis seems to be the principal feature and probable cause of tissue infarcts. Primary hippocampal cultures containing both neurons and astrocytes derived from fetal rats were used to examine the relative contributions of and interactions between excitotoxic and acidotic cell injury. Hypoxia-induced damage was energy dependent and involved the N-methyl-D-aspartate (NMDA) receptor. Glucose above 1 mM could completely protect against hypoxia-induced injury in a pH range of 7.4-6.5, while the NMDA receptor antagonist D,L-2-amino-5-phosphonovaleric acid (500 microM) during the posthypoxic period provided only partial protection in the absence of glucose. Astrocyte cultures were undamaged by ischemic-like treatment in this pH range, suggesting that hypoxia-induced cell death in mixed cultures was restricted to neurons. Lowering the extracellular pH to 7.0 and 6.5 caused no neuronal damage in normoxic controls, but in each case provided significant protection against hypoxic neuronal injury. In contrast, a second type of neurotoxicity was observed after a 6-h exposure to pH 6.0, while exposure to pH 5.5 was required to kill astrocytes. This acidotic damage appeared to be energy independent and did not involve the NMDA receptor. These results suggest that excitotoxic neuron death has an energetic component and that acidosis may produce both protective and damaging effects in the hippocampus during ischemic insults.

Hippocampal glutamine synthetase: insensitivity to glucocorticoids and stress.

Glucocorticoids enhance the neurotoxic potential of several insults to the rat hippocampus that involve overactivation of glutamatergic synapses. These hormones also stimulate the synthesis of glutamine synthetase (GS) in peripheral tissue. Because this enzyme helps regulate glutamate metabolism in the central nervous system, glucocorticoid induction of GS in the brain may underlie the observed synergy. We have measured GS activity in the hippocampus and skeletal muscle (plantaris) of adult rats after bilateral adrenalectomy (ADX), corticosterone (Cort) replacement, or stress. No significant changes in GS were observed in hippocampal tissue, whereas muscle GS was significantly elevated after Cort treatment or stress and was reduced after ADX. These results suggest that Cort-induced shifts in GS activity probably do not explain Cort neurotoxicity, although the stress-induced rise in muscle GS may be relevant to certain types of myopathy.

Mild acidosis protects hippocampal neurons from injury induced by oxygen and glucose deprivation.

Hippocampal neurons are extremely sensitive to ischemic injury; two plausible mechanisms have been implicated in mediating such damage. The first involves overexposure of neurons to excitatory N-methyl-D-aspartate (NMDA) receptor agonists, which mobilize damaging concentrations of intracellular calcium; the second involves the generation of damaging tissue acidosis. A recent report shows that exposure to pH 6.6 can block NMDA-induced calcium currents in hippocampal neurons. This suggests that moderate acidity might protect against NMDA-mediated neurotoxicity and ischemic injury in vivo. We have observed such projection in vitro using primary hippocampal cultures. At an extracellular pH of 7.4, 6 h of glucose-free anoxia caused delayed and profound damage to neurons which was partially attenuated by the NMDA receptor antagonist, 2-amino-5-phosphonovaleric acid (APV). Dropping the pH to 6.5 provided virtually complete protection against this insult. Thus, acidosis need not be viewed exclusively as a damaging component of ischemic insults.

Venn diagrams and neuronal vulnerability.

Greenamyre and Young note that there is poor correlation between which hippocampal regions are damaged in Alzheimer's disease and which have the highest concentrations of NMDA receptors. They conclude that EAAs can thus only be necessary, but not sufficient to explain Alzheimer's damage. We note that this is in fact probably the rule rather than the exception: some of the most credible agents which damage neurons are merely necessary, but not sufficient to explain selective neuronal vulnerability.

Stress and glucocorticoids in aging.

This article has considered two themes that have permeated the gerontologic literature--namely, that aging is a time of decreased efficiency in responding to stress and that chronic stress can accelerate aspects of aging. Given the restricted framework of considering adrenocortical function (as a component of the stress response) and glucocorticoid over-exposure (as a component of chronic stress), there is considerable evidence for both of these ideas. The capacity of glucocorticoids to damage the rat hippocampus slowly over the life span and the glucocorticoid hypersecretion that seems to ensue during aging as a result of such hippocampal damage support these long-standing ideas. It should be noted that these two components interact with each other--excessive glucocorticoid secretion damages the hippocampus, and hippocampal damage produces excessive glucocorticoid secretion. This dysregulatory cascade appears to be a normal part of aging in the rat. The role of glucocorticoids in triggering programmed aging and death, while quite dramatic, is probably a phylogenetically rare event; it remains to be seen if the dysregulatory cascade of glucocorticoid excess in the rat is of relevance to aging in other species. Numerous published studies suggest that this cascade is not an obligatory aspect of normal human aging; rather, it appears to be a significant factor in the explanation of some features of pathologies associated with human aging.