Alfred T. Malouf

Glycine site NMDA receptor antagonists provide protection against ischemia-induced neuronal damage in hippocampal slice cultures.

Ischemia-induced neuronal injury can be reduced by glutamate antagonists acting at the N-methyl-D-aspartate (NMDA) receptor. 7-Chlorokynurenic acid and the recently synthesized compound Acea 1021 block NMDA receptors by acting at the strychnine-insensitive glycine site. The anti-ischemic properties of these compounds were tested by evaluating their ability to reduce CA1 neuronal damage in hippocampal slice cultures deprived of oxygen and glucose. Acea 1021 and 7-chlorokynurenic acid significantly reduced CA1 injury produced by oxygen and glucose deprivation in a dose-dependent manner. The neuroprotective effect of these compounds was reversed by the addition of glycine. The phencyclidine site NMDA antagonist MK-801 also provided significant protection to CA1 neurons against the same insult, and this protection was not affected by the addition of glycine. These results indicate that Acea 1021 and 7-chlorokynurenic acid can provide protection to CA1 neurons against ischemia-induced injury by a glycine-sensitive mechanism.

Beta amyloid is neurotoxic in hippocampal slice cultures.

We examined the neurotoxicity of the 40 amino acid fragment of beta amyloid peptide (A beta 1-40) in cultured hippocampal slices. When injected into area CA3, A beta 1-40 produced widespread neuronal damage. Injection of the reverse sequence peptide, A beta 40-1, or vehicle alone produced little damage. The distribution A beta 1-40 was highly correlated with the area of neuronal damage. Thioflavine S and electron microscopic analysis confirmed that injected A beta 1-40 formed 7-9 nm AD type amyloid fibrils in the cultures. A beta 1-40 also altered the number of GFAP immunoreactive astrocytes and ED-1 immunoreactive microglia/macrophages within and around the A beta 1-40 deposit. The observed neurotoxicity of A beta 1-40 in hippocampal slice cultures provides evidence that this peptide may be responsible for the neurodegeneration observed in AD.

Effect of beta amyloid peptides on neurons in hippocampal slice cultures

Several investigators have described the neurotrophic and neurotoxic effects of beta amyloid peptide fragments on dissociated hippocampal neurons in culture. In these prior studies, the peptides were added to dissociated cultures between day 0 and day 4 in vitro, before hippocampal neurons are fully mature. We have analyzed the neurotrophic and neurotoxic effects of beta amyloid fragments beta 1-28, beta 25-35 and beta 1-40 on hippocampal slice cultures, whose physiology and morphology resembles the intact hippocampus. Addition of beta 1-28 or beta 25-35 to the growth medium did not produce significant changes in dendritic length or number of branches. Nerve growth factor, previously reported to enhance the neurotoxic effects of beta 1-40 on dissociated hippocampal neurons in culture, did not significantly enhance the neurotrophic effects of beta 1-28. To achieve high local concentrations of peptides and to avoid potential access problems in the cultures, we injected beta 1-28, beta 25-35, and beta 1-40 directly into the cultures. Amyloid-mediated neurotoxicity was not observed for beta 1-28 or beta 25-35, but beta 1-40 appeared to produce neurodegeneration around the site of injection.

Glutamate-mediated selective vulnerability to ischemia is present in organotypic cultures of hippocampus.

Ischemic damage to the brain, whether induced experimentally or observed clinically, often produces a pattern of delayed selective cell death in subfield CA1 of hippocampus which has been associated with significant neurologic deficits. The present study demonstrates that this selective vulnerability of CA1 neurons to ischemia, with relative preservation of their neighbors, is expressed in organotypic tissue culture and is prevented by the N-methyl-D-aspartate (NMDA) receptor blocker, MK-801. These data provide conclusive evidence that this selective cell death does not have a vascular etiology but is mediated by factors intrinsic to the hippocampal neurons and/or local circuitry. This model system provides an opportunity both to examine mechanisms of ischemic cell death in an avascular environment and to study methods of prevention in the absence of systemic variables.

Adenosinergic modulation of CA1 neuronal tolerance to glucose deprivation in organotypic hippocampal cultures

Glucose deprivation produced neuronal degeneration of CA1 pyramidal neurons in hippocampal slice cultures. The effects of the adenosine agonist cyclohexyladenosine (CHA) and antagonist cyclopentylxanthine (CPX) on CA1 neuronal loss following hypoglycemia was examined using propidium iodide fluorescence as an indicator of cell death. The intensity of propidium iodide fluorescence in hippocampal area CA1 was quantified using Optimas image analysis software. Following 2 or 3 h of glucose deprivation, CPX significantly enhanced injury in the CA1 region while CHA provided significant protection. These results suggest that adenosine plays an important role in endogenous neuronal protection during hypoglycemic injury, and also supports a role for the use of adenosine agonists as neuroprotective agents.

Glycine Causes Increased Excitability and Neurotoxicity by Activation of NMDA Receptors in the Hippocampus

Glycine is an inhibitory neurotransmitter in the spinal cord and also acts as a permissive cofactor required for activation of the N-methyl-D-aspartate (NMDA) receptor. We have found that high concentrations of glycine (10 mM) cause marked hyperexcitability and neurotoxicity in organotypic hippocampal slice cultures. The hyperexcitability, measured using intracellular recording in CA1 pyramidal neurons was completely blocked by the NMDA receptor antagonist MK-801 (10 microM), but not by the AMPA receptor antagonist DNQX (100 microM). The neurotoxicity caused by glycine occurred in all regions of hippocampal cultures but was most marked in area CA1. There was significant CA1 neuronal damage in cultures exposed to 10 mM glycine for 30 min or longer (P < 0.01) or those exposed to 4 mM glycine for 24 h compared to control cultures (P < 0.01). The NMDA antagonists MK-801 (10 microM) and APV (100 microM) significantly reduced glycine-induced neuronal damage in all hippocampal subfields (P < 0.01). The AMPA antagonists CNQX, DNQX, and NBQX (100 microM) had no effect on glycine-induced neuronal damage. High concentrations of glycine therefore appear to enhance the excitability of hippocampal slices in an NMDA receptor-dependent manner. The neurotoxic actions of glycine are also blocked by NMDA receptor antagonists.

Epileptiform activity in hippocampal slice cultures with normal inhibitory synaptic drive

The synaptic events responsible for epileptiform burst discharge are often difficult to define. Blockade of inhibition has been used to produce epileptiform events, but it is unclear whether increased excitatory activity in the presence of normal inhibition can also result in burst discharge. In the hippocampal slice culture preparation, a small percentage of cultures exhibit spontaneous bursts. To determine whether the absence of inhibitory postsynaptic potentials (IPSPs) is responsible for these spontaneous bursts, we applied the glutamate antagonist, kynurenic acid (KYN) to block burst activity, and unmask any underlying IPSPs. KYN (10 mM) quickly reduced synaptic activity with concomitant loss of burst discharge. Washout of KYN resulted in a gradual return of synaptic activity, during which time both fast and slow IPSPs were clearly observed. As burst activity returned to control levels, excitatory postsynaptic potentials (EPSPs) were increasingly superimposed within the inhibitory events, obscuring (but not eliminating) the IPSPs. In these hippocampal slice cultures, therefore, epileptiform bursts appear to be the result of an abnormally high level of excitatory synaptic drive, not a reduction in inhibition.

Differential effects of zinc on hyperpolarizing and depolarizing GABAA synaptic potentials in hippocampal slice cultures

We have examined the changes in GABAA-mediated synaptic potentials recorded from CA3 pyramidal neurons in hippocampal slice cultures following application of zinc (Zn2+). Unlike 4-AP, Zn2+ did not enhance fast hyperpolarizing potentials but primarily enhanced depolarizing GABAA potentials. Zn2+ did not alter the postsynaptic response of pyramidal neurons to pressure applied GABA, consistent with previous reports that Zn2+ enhances the release of GABA from presynaptic terminals. To examine the role of local circuitry in the production of Zn2+ responses, we recorded from cultures maintained for 7-10 days following removal of the dentate and hilus to allow complete degeneration of the mossy fibers (DGX cultures). Zn2+ produced giant depolarizing potentials (GDPs) in DGX cultures that were identical to those in intact cultures. In contrast, the 4-AP response was dramatically altered in DGX cultures. In DGX cultures, Zn2+ co-applied with 4-AP appeared to inhibit the production of fast hyperpolarizing GABAA synaptic potentials produced by 4-AP alone. This inhibition of fast hyperpolarizing potentials suggests that Zn2+ may reduce the release of GABA onto pyramidal cell somata. These observations suggest that Zn2+ enhances GABA release from local circuit neurons that synapse onto pyramidal cell dendrites, and inhibits GABA release onto pyramidal cell somata.

Slice cultures of the imprinting-relevant forebrain area medio-rostral neostriatum/hyperstriatum ventrale of the domestic chick: immunocytochemical characterization of neurons containing Ca2+-binding proteins

The forebrain area medio-rostral neostriatum/hyperstriatum ventrale, a presumed analogue to the mammalian prefrontal cortex, displays a variety of synaptic changes during auditory filial imprinting. In order to study the underlying basic mechanisms of this synaptic plasticity we developed slice cultures of the medio-rostral neostriatum/hyperstriatum ventrale from newly hatched chicks. As a prerequisite for these investigations and in order to test the suitability of this system for future studies, we performed a thorough characterization of the in vitro tissue, of its cellular components and some of their biochemical features in comparison with in situ tissue. Since in situ the medio-rostral neostriatum/hyperstriatum ventrale has been previously shown to contain three distinct neuron populations characterized by the activity-regulated Ca(2+)-binding proteins parvalbumin, calbindin D28K and calretinin, we used these proteins as neuronal markers to study the survival and preservation of the morphological features of medio-rostral neostriatum/hyperstriatum ventrale neurons in vitro. In agreement with in vivo studies the three Ca(2+)-binding proteins are confined to neuronal cells and they are not colocalized, i.e. they appear to characterize three different neuron populations. The immunoreactive neurons in medio-rostral neostriatum/hyperstriatum ventrale cultures to a certain extent appear to form synaptic contacts with each other, shown by the double immuncytochemical experiments. One difference between cells in vivo and in vitro is their soma size, which is much larger in vitro than in vivo. This and our previous study on neuronal morphology demonstrates that morphologically and biochemically intact neurons can be maintained in medio-rostral neostriatum/hyperstriatum ventrale slice cultures, which may thus provide a suitable in vitro system for further studies of neuronal and synaptic plasticity in vitro.