Maurizio Balestrino

Block of (Na+,K+)ATPase with ouabain induces spreading depression-like depolarization in hippocampal slices

We used ouabain (100 microM) to block Na+,K(+)ATPase of in vitro rat hippocampal slices. This treatment was sufficient to cause the sudden depolarization that is the hallmark of both spreading depression (SD) and of the SD-like anoxic depolarization (AD). This depolarization was accompanied by a large and sudden increase in [K](o), also reminiscent of that observed during both SD and AD. Ouabain-induced SD did not require a complete inactivation of Na+,K(+)ATPase, as it occurred when the enzyme was still capable of providing recovery of both V(o) and [K](o). The data indicate that functional inactivation of Na+,K(+)ATPase per se initiates events that lead to an SD-like AD. This ouabain-induced depolarization was not affected by block of synaptic transmission, instead it was abolished by hyperosmolarity of the extracellular space. The possible relevance of these findings to the pathophysiology of AD is discussed.

Chlorpromazine protects brain tissue in hypoxia by delaying spreading depression-mediated calcium influx

We have investigated the possible protective effect of chlorpromazine in hypoxia of brain tissue, using rat hippocampal slices maintained at 35-36 degrees C. The recovery of synaptic transmission along the Schaffer collaterals to the CA1 pathway after 9 min hypoxia was compared in chlorpromazine-treated and in control slices. Recovery upon reoxygenation was the exception in control slices, while it was observed in approximately 50 and 100% of slices treated with 7 and 70 microM chlorpromazine, respectively. Chlorpromazine also significantly delayed the occurrence of the hypoxia-induced spreading depression (SD). Recovery took place when SD occurred late during hypoxia, not when it occurred early. In those slices in which 7 microM chlorpromazine afforded no protection, SD occurred as early as it did in control slices. In further experiments, we deliberately induced SD during hypoxia in 70 microM-treated slices by topically applying a drop of high-K+ artificial cerebrospinal fluid (ACSF). Recovery was not observed when SD was induced early, but it was observed when it was induced near the end of the hypoxic period. Slices exposed to the same period of hypoxia in Ca2+-free ACSF recovered synaptic transmission (even without chlorpromazine treatment) despite early induction of SD. We conclude that: chlorpromazine protects brain tissue from hypoxia-induced irreversible loss of synaptic transmission; it does so by delaying the occurrence of SD, and hence shortening the time spent in the SD-induced depolarized state; and the harm done by SD in hypoxia is related to the influx of Ca2+ into neurons.

Spreading depression-like hypoxic depolarization in CA1 and fascia dentata of hippocampal slices: relationship to selective vulnerability

Hippocampal tissue slices were made hypoxic for 4-10 min and then reoxygenated for 60-120 min. Postsynaptic evoked potentials were recorded and extracellular DC potential was monitored continuously in stratum (st.) pyramidale of CA1 and st. granulosum of fascia dentata (FD). In some preparations extracellular potassium ([K+]o) and calcium ([Ca2+]o) were also recorded in both regions. Postsynaptic responses disappeared sooner during hypoxia and were less likely to recover upon reoxygenation in CA1 than in FD. The CA1 region exhibited a spreading depression (SD)-like response to hypoxia more often than did FD. When both regions showed SD-like depolarization, voltage shift and elevation of [K+]o were of greater magnitude and shorter latency in CA1. The probability of posthypoxic recovery of synaptic transmission was inversely related to the time spent in the SD-like state in both CA1 and FD. We conclude that the selective vulnerability of CA1 neurons to hypoxic and ischemic damage may be due, at least in part, to the regions propensity to undergo prolonged and severe SD-like depolarization.

Exogenous creatine delays anoxic depolarization and protects from hypoxic damage: dose–effect relationship

Incubation of hippocampal slices with different concentrations of creatine (0.5, 1, 10, 25 mM) results in a dose-dependent increase in intracellular phosphocreatine (PCr). Electrophysiological evidence suggests that this effect can protect neurons from anoxic damage by delaying the depletion of ATP during oxygen deprivation. In this paper we show that incubation of brain slices with varying doses of creatine increases intracellular phosphocreatine and delays anoxic depolarization (AD) in a dose-dependent way. Specifically, addition to the incubation medium of 1 mM creatine significantly increased AD latency during hypoxia and prevented irreversible neuronal damage. Adding 0.5 mM creatine had no significant effect. Higher concentrations of creatine (up to 25 mM) did not provide any better protection. Our data also suggest a linear correlation between intracellular PCr and AD latency. These data report neural protection by exogenous creatine at concentrations lower than those usually reported in the literature.

The effects of moderate changes of extracellular K+ and Ca2+ on synaptic and neural function in the CA1 region of the hippocampal slice

The effects of moderate changes of the concentration of ions on the function of mammalian central nervous tissue have not exactly been determined. We placed tissue slices from rat hippocampal formation in an interface chamber for study in vitro. Extracellular potentials were recorded in stratum radiatum and stratum pyramidale in response to stimuli of varying intensity applied to the Schaffer collateral bundle. The overall input-output relationship of excitatory synaptic transmission was gauged by expressing postsynaptic population spike amplitude as a function of presynaptic volley amplitude. The components of the transmission process were also examined by plotting the maximal rate of rise (slope) of the focally recorded synaptic potential (fEPSP) as a function of presynaptic volley amplitude, and the population spike amplitude as a function of the fEPSP slope. Raising the concentration of K+ from the normal level of 3.5 mM to 5 mM caused an average increase of 48% in the population spike evoked by a given presynaptic volley. This was due to an increased electrical excitability of pyramidal cells, as indicated by an increase of the population spike evoked by a given magnitude of fEPSP. Conversely, lowering [K+]o from 3.5 to 2 mM caused a decrease of the population spike relative to a given magnitude of either the presynaptic volley or the fEPSP. Changing [K+]o within these limits caused no significant change of the fEPSP evoked by a given presynaptic volley. Raising [Ca2+]o from 1.2 to 1.8 mM caused a 35% increase in both the fEPSP and the population spike evoked by a given presynaptic volley, and lowering [Ca2+]o to 0.8 mM caused a decrease of both these functions. The amplitude of the population spikes evoked by given fEPSPs changed surprisingly little (but consistently) when [Ca2+]o was varied within these limits. We conclude that moderate changes of [K+]o influence mainly the electric excitability of hippocampal pyramidal cells, with little effect on transmitter release or on the response of the postsynaptic membrane to transmitter, while moderate changes of [Ca2+]o affect the release of excitatory synaptic transmitter more than they affect postsynaptic membrane function.

NMDA antagonists: Lack of protective effect against hypoxic damage in CA1 region of hippocampal slices

Rat hippocampal slices were exposed to a hypoxic insult in control medium or while exposed to the N-methyl-D-aspartate (NMDA) receptor antagonists DL-2-amino-7-phosphonoheptanoic acid (100 microM) or DL-2-amino-5-phosphonovaleric acid (25 or 100 microM). Synaptic transmission between Schaffer collaterals and CA 1 pyramidal cells was evaluated before and after the hypoxic period, and the DC potential in the CA 1 pyramidal cell layer was monitored during the hypoxic period. Neither antagonist significantly increased the proportion of slices in which synaptic transmission recovered following hypoxia, nor did they increase the latency of the spreading depression-like anoxic depolarization. We suggest that NMDA receptors are not involved in neural damage caused by severe hypoxia with sudden onset, while they may play a role in the effects of more moderate, gradual onset hypoxia and in post-ischemic reperfusion effects.

An array of Pt-tip microelectrodes for extracellular monitoring of activity of brain slices

A microelectrode array (MEA) consisting of 34 silicon nitride passivated Pt-tip microelectrodes embedded on a perforated silicon substrate (porosity 35%) has been realized. The electrodes are 47 microns high, of which only the top 15 microns are exposed Pt-tips having a curvature of 0.5 micron. The MEA is intended for extracellular recordings of brain slices in vitro. Here we report the fabrication, characterization and initial electrophysiological evaluation of the first generation of Pt-tip MEAs.

Electrophoretic characterization of gold nanoparticles functionalized with human serum albumin (HSA) and creatine

The synthesis of composite nanoparticles consisting of a gold core coated with a human serum albumin (HSA)/creatine layer is described, and their possible application as novel drug carriers for brain delivery is discussed. In this paper, the effect of the concentration of creatine and HSA in the different formulations is studied by electrophoretic mobility measurements as a function of pH and ionic strength. Due to the permeable character of the coatings surrounding the gold cores, an appropriate analysis of their electrophoretic mobility must be addressed. Recent developments of electrokinetic theories for particles covered by soft surface layers have rendered possible the evaluation of the softness degree from raw electrophoretic mobility data. In the present contribution, the data are quantitatively analyzed on the basis of three theoretical models of the electrokinetics of soft particles. As a result, information is obtained on both the surface potential and the charge density of the surrounding layer. The three models used reproduce properly the experimental behavior, although Duval and Ohshimas calculations appear to yield a more accurate fit of the data. It is shown that the albumin/nanogold particles absorb large amounts of creatine. In addition, the low surface charge and the albumin layer are expected to make it possible to deliver the particles through the blood-brain barrier.

The Role of Spreading Depression-Like Hypoxic Depolarization in Irreversible Neuron Damage, and its Prevention

We investigated the role of spreading depression (SD)-like depolarization in hypoxic neuron damage. In hippocampal tissue slices, drugs that delayed the onset of the SD-like depolarization triggered by hypoxia (and therefore shortened the time spent in SD) also improved the chances of recovery of neural function after reoxygenation. The protective effect and the delay of SD onset were linked: in the few cases when SD was not delayed, neuronal function did not recover. By contrast, if SD-like depolarization was provoked (by high K+) early during hypoxia, functional recovery did not occur in spite of the presence of a protective drug. Dentate granule cells recovered function more frequently than did CA1 pyramidal cells, and during hypoxia SD-like depolarization began later and was milder in fascia dentata (FD) than in CA1 sector of hippocampal tissue slices. SD-induced damage was dependent on the availability of extracellular calcium: if Ca2+ was withdrawn before and during a hypoxic episode, then synaptic function recovered even after SD of extended duration. Iontophoretic injection of Ca2+ but not of Mg2+ into giant neurons of Aplysia caused irreversible loss of electric excitability and membrane impedance. We conclude that prolonged SD-like depolarization injures neurons because it allows excessive intracellular accumulation of calcium. We argue that SD is one, but not the only, mechanism by which hypoxic/ischemic neurons can be injured, and we advocate a multi-pronged approach to clinical management.

Electrophysiological effects of sustained delivery of CRF and its receptor agonists in hippocampal slices.

The corticotropin-releasing factor (CRF) is a hypothalamic peptide that regulates the release of adrenocorticotropic hormone (ATCH) and of beta-endorphin. It has been suggested that it modulates learning and memory processes in rat. However, the electrophysiological effects that CRF produces on hippocampal neurons have been so far little investigated. In particular, the effects of CRF on long-term potentiation (LTP), a phenomenon which is thought to be the substrate of memory processes, are unknown. We studied the effects of sustained administration of CRF and of two of its receptor agonists on basal neuronal activity and on in vitro hippocampal LTP. The two receptor agonists were D-Glu-20-CRF and D-Pro-5-CRF, selective for the CRF-R1 and the CRF-R2 receptors, respectively. We found that CRF, D-Pro-5-CRF and D-Glu-20-CRF at the concentration of 1 nM diminish the amplitude of hippocampal population spike and prevent the onset of LTP. Higher concentrations of CFR have less depressing effects on neuronal activity, yet they still prevent the occurrence of LTP.