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.

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.

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.

Involvement of S-100 protein in anoxic long-term potentiation.

In in vitro rat hippocampal slices a short period (2 min) of hypoxia resulted in lasting potentiation of the population spike transynaptically evoked in CA1 by stimulation of Schaffer collaterals ("anoxic LTP"). Pretreatment of slices with antiserum against S-100 protein fully prevented this anoxic LTP. Since also "classical" (i.e., induced by high-frequency electrical stimulation) long-term potentiation is prevented by anti S-100 serum, this represents one more important similarity between these events.

Antiserum against S-100 protein prevents long term potentiation through a cAMP-related mechanism.

Long term potentiation (LTP) was induced in the CA1 region of rat hippocampal slices by tetanization of the Schaffer collaterals. Local pretreatment of CA1 with serum of rabbits immunized against S-100 prevented the potentiation. However, treatment of the slices with a membrane permeant cAMP analogue, such as 8-Br-cAMP, could protect against the blocking effect of anti S-100 serum. We suggest that in the rat endogenous S-100b is involved in transduction mechanisms during LTP induction, via its ability to stimulate adenylate cyclase. Possible mechanisms of this action are discussed.

Role of creatine and phosphocreatine in neuronal protection from anoxic and ischemic damage.

Phosphocreatine can to some extent compensate for the lack of ATP synthesis that is caused in the brain by deprivation of oxygen or glucose. Treatment of in vitro rat hippocampal slices with creatine increases the neuronal store of phosphocreatine. In this way it increases the resistance of the tissue to anoxic or ischemic damage. In in vitro brain slices pretreatment with creatine delays anoxic depolarization (AD) and prevents the irreversible loss of evoked potentials that is caused by transient anoxia, although it seems so far not to be active against milder, not AD-mediated, damage. Although creatine crosses poorly the blood-brain barrier, its administration in vivo at high doses through the intracerebroventricular or the intraperitoneal way causes an increase of cerebral phosphocreatine that has been shown to be of therapeutic value in vitro. Accordingly, preliminary data show that creatine pretreatment decreases ischemic damage in vivo.

Increase of cerebral phosphocreatine in normal rats after intracerebroventricular administration of creatine.

Intracerebroventricular (ICV) administration of creatine increased cerebral phosphocreatine in normal rats by 67%, the highest increase so far reported in an in vivo model. We used osmotic minipumps (Alzet, Palo Alto, CA, USA) to administer creatine, 0.5 mM, to the lateral ventricle at the rate of 10 microl/h for 3 days. Brain phosphocreatine in saline-treated controls was 33 +/- 17 microM/g protein (mean +/- SD, N = 9). In creatine-treated rats (0.5 mM for 3 days) such content was 55 +/- 17 microM/g protein (mean +/- SD, N = 7). This difference is statistically significant (p = 0.02, t-test). The increase we found in cerebral phosphocreatine is of an order of magnitude comparable to the increase previously found in in vitro experiments, and may be effective in protecting brain tissue from ischemic damage.