Roland Willems

Evidence for a role of the N-methyl-d-aspartate (NMDA) receptor in cortical spreading depression in the rat

The neurotransmitter glutamate activates the N-methyl-D-aspartate (NMDA), quisqualate and kainate receptors. It has been proposed, but also disputed, that local release of glutamate would play a pivotal role in cortical spreading depression (SD). We tested this hypothesis by investigating the influence of NMDA antagonists on SD, using the non-competitive NMDA antagonists ketamine, phencyclidine (PCP) and MK-801 and the competitive NMDA antagonist DL-2-amino-7-phosphonoheptanoate (2-APH), injected intraperitoneally in rats anesthetized with alfentanil. SD was elicited by cathodal DC-stimulation of the frontal cortex. SD propagation was followed using two ion-sensitive microelectrodes placed in the parietal and occipital cortex. The NMDA antagonists increased SD threshold, decreased the propagation velocity and decreased the duration of the accompanying extracellular DC, K+ and Ca2+ changes at the following doses: 40 mg/kg ketamine, 10 mg/kg PCP, 0.63 mg/kg MK-801, 10 and 40 mg/kg 2-APH. With each NMDA antagonist failure of SD propagation between both microelectrodes could be observed. SD elicitation (or propagation) was inhibited completely with 80 mg/kg ketamine, 3.1 mg/kg MK-801 and 160 mg/kg 2-APH. These NMDA antagonists have also anticonvulsant properties. None of these effects on SD were observed with high doses of other anticonvulsants such as 80 mg/kg phenytoin or 40 mg/kg diazepam. These experiments indicate that endogenous release of excitatory amino acids and their action on the NMDA receptor play an important role in the initiation, propagation and duration of SD.

Dorsal-ventral gradient in vulnerability of CA1 hippocampus to ischemia: a combined histological and electrophysiological study.

Transverse hippocampal slices were prepared after 7 days survival from rats subjected to 8 min of global incomplete ischemia by temporary occlusion of both carotid arteries and hypotension. The slices demonstrated a dorsal-ventral gradient in the amount of ischemic neuronal necrosis in the CA1 region. Histologically ischemic cell change decreased from 90% dorsoseptally to 10% ventrotemporally. Electrophysiological analysis of the number of slices with viable synaptic transmission in CA1 also revealed a septotemporal gradient in susceptibility to ischemia.

Altered Na+-channel function as an in vitro model of the ischemic penumbra: action of lubeluzole and other neuroprotective drugs

Veratridine blocks Na(+)-channel inactivation and causes a persistant Na(+)-influx. Exposure of hippocampal slices to 10 microM veratridine led to a failure of synaptic transmission, repetitive spreading depression (SD)-like depolarizations of increasing duration, loss of Ca(+)-homeostasis, a large reduction of membrane potential, spongious edema and metabolic failure. Normalization of the amplitude of the negative DC shift evoked by high K+ ACSF 80 min after veratridine exposure was taken as the primary endpoint for neuroprotection. Compounds whose mechanisms of action includes Na(+)-channel modulation were neuroprotective (IC50-values in microM): tetrodotoxin 0.017, verapamil 1.18, riluzole 1.95, lamotrigine > or = 10, and diphenylhydantoin 16.1. Both NMDA (MK-801 and PH) and non-NMDA (NBQX) excitatory amino acid antagonists were inactive, as were NOS-synthesis inhibitor (nitro-L-arginine and L-NAME) Ca(2+)-channel blockers (cadmium, nimodipine) and a K(+)-channel blocker (TEA). Lubeluzole significantly delayed in time before the slices became epileptic, postponed the first SD-like depolarization, allowed the slices to better recover their membrane potential after a larger number of SD-like DC depolarizations, preserved Ca2+ and energy homeostasis, and prevented the neurotoxic effects of veratridine (IC50-value 0.54 microM). A concentration of lubeluzole, which was 40 x higher than its IC50-value for neuroprotection against veratridine, had no effect on repetitive Na(+)-dependent action potentials induced by depolarizing current in normal ACSF. The ability of lubeluzole to prevent the pathological consequences of excessive Na(+)-influx, without altering normal Na(+)- channel function may be of benefit in stroke.

Hypolocomotive behaviour associated with increased microglia in a prenatal immune activation model with relevance to schizophrenia

Over the past decade a neurodevelopmental animal model with high validity for schizophrenia has been developed based on the environmental risk factor known as maternal immune activation (MIA). The immunological basis of this model, together with extensive data from clinical and preclinical context, suggests the involvement of an aberrant neuro-immune system in the pathophysiology of schizophrenia. The goal of this study was to examine microglia activation in adult behaviourally phenotyped MIA offspring. MIA was induced in pregnant rats using viral mimetic Poly I:C at gestational day 15. Adult offspring were behaviourally phenotyped at postnatal days (PND) 56, 90 and 180 through the evaluation of prepulse inhibition (PPI) of the acoustic startle and spontaneous locomotion. Finally, the presence of activated microglia in brain regions associated with schizophrenia was evaluated using post-mortem immunohistochemistry against OX-42 (CD11b) and ED-1 (CD68). Although a deficit in PPI could not be replicated despite the high number of animals tested, we found an overall decrease in basal startle response and spontaneous locomotion in offspring born to Poly I:C- compared to saline-treated dams, accompanied by increased microglial density with characteristics of non-reactive activation in the chronic stage of the model. These findings provide additional evidence for a role played by microglial activation in schizophrenia-related pathology in general and psychomotor slowing in particular, and warrant extensive research on the underlying mechanism in order to establish new drug targets for the treatment of schizophrenia patients with an inflammatory component.

Extracellular ions during veratridine-induced neurotoxicity in hippocampal slices: neuroprotective effects of flunarizine and tetrodotoxin

Veratridine, by blocking Na+ channel inactivation and shifting activation to more negative membrane potentials, causes Na(+)-influx and a persistent tendency for depolarization. Veratridine is neurotoxic to cultured neurones, and this neurotoxicity can be blocked by the class IV calcium antagonist, flunarizine. We were interested to know whether similar effects could be found in a functional differentiated tissue containing adult neurones and glial cells. We examined this in hippocampal slices using extracellular potential recordings and ion-selective microelectrodes sensitive to [Na+]o, [Ca2+]o and [K+]o. Veratridine blocked synaptic transmission in CA1, and induced several episodes of spreading depression (SD). This was followed by a long-lasting increase in [K+]o and a continuous decrease in [Ca+]o. Following veratridine exposure to hypoxia only revealed a small negative DC shift and small shifts in extracellular ions; indicating that the cells had lost the ability to maintain ion homeostasis before the hypoxia, and that veratridine had been neurotoxic. In hippocampal slices obtained from guinea pigs which had been pretreated with 40 mg/kg x 2 flunarizine orally the time before the first SD induced by veratridine was doubled. Although the ion shifts during the first SD were similar to controls, flunarizine reduced the time of recovery of [Ca2+]o, [K+]o and DC potential. The increase in [K+]o baseline and the massive decrease in [Ca2+]o baseline seen following the SDs in the solvent group were smaller in the flunarizine-treated slices. During the subsequent hypoxic period the negative DC shift was 8x larger in the flunarizine group, and the shifts in [K+]o, [Na+]o and [Ca2+]o were bigger. Tetrodotoxin also delayed the first SD during veratridine and increased the size of the DC shift during the subsequent hypoxic period. Both flunarizine and tetrodotoxin therefore protected adult brain tissue containing glia from the neurotoxicity of veratridine. These findings suggest that persistent Na(+)-influx and the consequent Ca2(+)-influx produce neurotoxicity, and that the ability to attenuate this neurotoxicity may be important in the mechanism of action of cerebroprotective drugs from different pharmacological classes.

NMDA Antagonists Inhibit Cortical Spreading Depression, But Accelerate the Onset of Neuronal Depolarization Induced by Asphyxia

Cortical spreading depression (SD) is a transient massive change of the local cortical microenvironment, which spreads over the cortex at a rate of approximately 3 mm/min. Asphyxia can facilitate the elicitation of SD. It has been proposed that release of glutamate may play an important role in SD1 and hypoxia. Glutamate activates the N-methyl-D-aspartate (NMDA), quisqualate and kainate receptors. We investigated whether the NMDA receptor is involved in SD during normoxia using the non-competitive NMDA antagonists ketamine, phencyclidine and MK-801 and the competitive NMDA antagonist 2-amino-7-phosphonoheptanoate (2-APH). Additionally, we tested whether NMDA antagonists affect the DC and ionic changes during asphyxia.

N-methyl-d-aspartate and hypoxia induced Ca2+-changes in the CA1 region of the hippocampal slice

Hypoxic neuronal depolarization was accompanied by a large decrease in extracellular [Ca2+]. After reoxygenation, the time at which [Ca2+] normalized was correlated with the extent of recovery of N-methyl-D-aspartate (NMDA) and synaptic responses. There was no evidence that the NMDA receptor system was more disrupted following hypoxia than the receptors involved in synaptic transmission. The Na+/K+ pump appeared to be better able to recover from hypoxia than the NMDA responses or synaptic transmission.

Field‐Potential Assay of Antiepileptic Drugs in the Hippocampal Slice

Summary: The effects of nine clinically active antiepileptic drugs and the NMDA antagonist 2‐amino‐7‐phosphonoheptanoic acid (2‐APH) were examined in three models in the in vitro hippocampal slice. In the “low Mg2 +” model, removal of Mg2+ from the perfusion fluid increased excitatory neurotransmission and led to epileptogenic field potentials. In the “low Ca2 +” model, decrease of Ca2 + and increase of Mg2 + and K+ in the perfusion fluid induced spontaneous “bursts” in the absence of synaptic transmission. Paired‐pulse stimulation was used to estimate the strength of recurrent inhibition in the “inhibition” model. The rank order of the potency of the compounds to antagonize the second epileptogenic population spike in the low Mg2+ model was 2‐APH > pentobarbital > midazolam > phenytoin > carbamazepine > chlordiazepoxide > phenobarbital = fluraze‐pam. Ethosuximide and valproate were inactive. In the low Ca2+ model, the rank order of the potency of the drugs to antagonize spontaneous epileptogenic bursts was phenytoin > carbamazepine > midazolam > pentobarbital > chlordiazepoxide > flurazepam > phenobarbital. 2‐APH, ethosuximide, and valproate were inactive. Only pentobarbital was active in the inhibition model. These experiments demonstrate the potential of in vitro tests in the hippocampus to reveal profiles of anticonvulsant activity.

Selective vulnerability of synaptic transmission in hippocampus to ex-vivo ischemia: effects of extracellular ionic substitution in the postischemic period

After 10-60 min of normothermic complete ischemia, hippocampal slices were prepared and allowed to recover for 60 min. The presence or absence of an evoked transsynaptic response was measured in CA1, CA3, and dentate gyrus. A selective vulnerability of the field excitatory postsynaptic potential to ischemia was found (CA1 greater than CA3 greater than dentate gyrus). Recovery of synaptic transmission in CA1 and CA3 was significantly improved by decreasing extracellular Ca2+ and increasing Mg2+ after ischemia. Addition of an N-methyl-D-aspartate antagonist further improved functional recovery. Postischemic reduction in extracellular Cl- increased recovery in CA1 and CA3, whilst reduction in Na+ was deleterious.

Subchronic memantine induced concurrent functional disconnectivity and altered ultra-structural tissue integrity in the rodent brain: revealed by multimodal MRI

BackgroundAn effective NMDA antagonist imaging model may find key utility in advancing schizophrenia drug discovery research. We investigated effects of subchronic treatment with the NMDA antagonist memantine by using behavioural observation and multimodal MRI.MethodsPharmacological MRI (phMRI) was used to map the neuroanatomical binding sites of memantine after acute and subchronic treatment. Resting state fMRI (rs-fMRI) and diffusion MRI were used to study the changes in functional connectivity (FC) and ultra-structural tissue integrity before and after subchronic memantine treatment. Further corroborating behavioural evidences were documented.ResultsDose-dependent phMRI activation was observed in the prelimbic cortex following acute doses of memantine. Subchronic treatment revealed significant effects in the hippocampus, cingulate, prelimbic and retrosplenial cortices. Decreases in FC amongst the hippocampal and frontal cortical structures (prelimbic, cingulate) were apparent through rs-fMRI investigation, indicating a loss of connectivity. Diffusion kurtosis MRI showed decreases in fractional anisotropy and mean diffusivity changes, suggesting ultra-structural changes in the hippocampus and cingulate cortex. Limited behavioural assessment suggested that memantine induced behavioural effects comparable to other NMDA antagonists as measured by locomotor hyperactivity and that the effects could be reversed by antipsychotic drugs.ConclusionOur findings substantiate the hypothesis that repeated NMDA receptor blockade with nonspecific, noncompetitive NMDA antagonists may lead to functional and ultra-structural alterations, particularly in the hippocampus and cingulate cortex. These changes may underlie the behavioural effects. Furthermore, the present findings underscore the utility and the translational potential of multimodal MR imaging and acute/subchronic memantine model in the search for novel disease-modifying treatments for schizophrenia.