A. Cupello

Nitric oxide and GABAA receptor function in the rat cerebral cortex and cerebellar granule cells.

The aim of the present work was to investigate the mechanism by which the diffusible factor nitric oxide regulates GABAA receptor function in the brain. The effect of nitric oxide on GABAA receptor function has been studied in two different neuronal preparations: rat cerebral cortex microsacs and rat cerebellum granule cells in culture. In the first case, GABA-stimulated 36Cl-accumulation was studied as an index of GABAA receptor function. The maximal rate of GABA-stimulated 36Cl- accumulation (Vmax) was reduced by treatment of microsacs with nitric oxide chemical donors such as sodium nitroprusside (-26%) and S-nitroso-acetyl-penicillamine (-11%). The greater effect of the former agent is due to an additional interference by its breakdown products. The biochemical precursor L-arginine (1 mM) produced the same Vmax decrease as S-nitroso-acetyl-penicillamine. This effect was reversed by a nitric oxide synthase blocker and appears truly nitric oxide mediated. The action of nitric oxide in this system does not seem to imply cyclic GMP formation. GABAA receptor function was studied by whole-cell patch-clamp in rat cerebellum granule cells in culture. In this case, L-arginine (100 microM) profoundly reduced the Cl- current elicited by 10 microM GABA and its effect subsided following washing out. The effect of L-arginine was observed almost exclusively on the rapidly desensitizing component of the GABA-activated current. The action of L-arginine was blocked by a protein kinase G inhibitor and mimicked by its activators. Thus, it appears that this effect in these cells involves nitric oxide formation, cyclic GMP accumulation and protein kinase G-catalysed phosphorylation of GABAA receptor.

Modulation by nitric oxide of rat brain GABAA receptors

The effect of nitric oxide (NO) on the function of GABAA receptors was studied in two different rat brain neuron populations. Cerebral cortex neuronal GABAA receptors were studied by preparing microsacs and evaluating 36Cl- accumulation. Whether nitric oxide was provided by sodium nitroprusside (SNP) or by the metabolic precursor precursor arginine there was a 15-25% reduction in the Vmax for GABA-stimulated 36Cl- accumulation. The arginine effect could be reversed by the NO synthase (NOS) inhibitor N omega-nitro-L-arginine. GABAA receptor mediated Cl- currents were studied in rat cerebellar granule cells by whole-cell patch clamp. S-Nitroso-N-acetylpenicillamine (SNAP), sodium nitroprusside and L-arginine reduced the Cl- current elicited by 10 microM GABA. The L-arginine effect was reversible upon its washing out. This circumstance indicates that NO produced by endogenous NOS can inhibit GABAA receptor function in cerebellar granule cells.

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

Summary. 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.

The creatine transporter mediates the uptake of creatine by brain tissue, but not the uptake of two creatine-derived compounds

Hereditary creatine transporter deficiency causes brain damage, despite the brain having the enzymes to synthesize creatine. Such damage occurring despite an endogenous synthesis is not easily explained. This condition is incurable, because creatine may not be delivered to the brain without its transporter. Creatine-derived compounds that crossed the blood-brain barrier in a transporter-independent fashion would be useful in the therapy of hereditary creatine transporter deficiency, and possibly also in neuroprotection against brain anoxia or ischemia. We tested the double hypothesis that: (1) the creatine carrier is needed to make creatine cross the plasma membrane of brain cells and (2) creatine-derived molecules may cross this plasma membrane independently of the creatine carrier. In in vitro mouse hippocampal slices, incubation with creatine increased creatine and phosphocreatine content of the tissue. Inhibition of the creatine transporter with 3-guanidinopropionic acid (GPA) dose-dependently prevented this increase. Incubation with creatine benzyl ester (CrOBzl) or phosphocreatine-Mg-complex acetate (PCr-Mg-CPLX) increased tissue creatine content, not phosphocreatine. This increase was not prevented by GPA. Thus, the creatine transporter is required for creatine uptake through the plasma membrane. Since there is a strong indication that creatine in the brain is mainly synthesized by glial cells and transferred to neurons, this might explain why hereditary transporter deficiency is attended by severe brain damage despite the possibility of an endogenous synthesis. CrOBzl and PCr-Mg-CPLX cross the plasma membrane in a transporter-independent way, and might be useful in the therapy of hereditary creatine transporter deficiency. They may also prove useful in the therapy of brain anoxia or ischemia.

Successful Treatment of Epilepsy with Serotonin Reuptake Inhibitors: Proposed Mechanism

The widely used antidepressants Specific Serotonin Reuptake Inhibitors (SSRI) have been tried with success as anticonvulsants in cases of nonsymptomatic epilepsy. This attempt was performed on the basis of experimental data suggesting the involvement of impairments of the serotonin system in the genesis of epilepsy. This overview summarizes the clinical data and presents biochemical and neurochemical evidences suggesting the mechanism of the therapeutic effects of SSRI in nonsymptomatic epilepsy. In particular, studies on blood-borne neutral amino acids and platelet serotonin transporter (SERT) in epileptics suggest: (a) That a decreased brain availability of tryptophan may be related to some types of epilepsy. (b) That reduction of the density of SERT may be a homeostatic reaction in the brain following epileptic seizures.

A dual mechanism for impairment of GABAA receptor activity by NMDA receptor activation in rat cerebellum granule cells

Abstract The function of the GABAA receptor has been studied using the whole cell voltage clamp recording technique in rat cerebellum granule cells in culture. Activation of NMDA-type glutamate receptors causes a reduction in the effect of GABA. Full GABAA receptor activity was recovered after washing out NMDA and NMDA action was prevented in a Mg++ containing medium. The NMDA effect was also absent when extracellular Ca++ was replaced by Ba++ and when 10 mM Bapta was present in the intracellular solution. Charge accumulations via voltage activated Ca++ channels greater than the ones via NMDA receptors do not cause any reduction in GABAA receptor function, suggesting that Ca++ influx through NMDA receptor channels is critical for the effect. The NMDA effect was reduced by including adenosine-5′-O-3-thiophosphate (ATP-γ-S) in the internal solution and there was a reduction in the NMDA effect caused by deltamethrin, a calcineurin inhibitor. Part of the NMDA induced GABAA receptor impairment was prevented by prior treatment with L-arginine. Analogously, part of the NMDA effect was prevented by blockage of NO-synthase activity by Nω-nitro-L-arginine. A combination of NO-synthase and calcineurin inhibitors completely eliminated the NMDA action. An analogous result was obtained by combining the NO-synthase inhibitor with the addition of ATP-γ-S to the pipette medium. The additivity of the prevention of the NMDA impairment of GABAA receptor by blocking the L-arginine/NO pathway and inhibiting calcineurin activity suggests an independent involvement of these two pathways in the interaction between NMDA and the GABAA receptor. On the one hand Ca++ influx across NMDA channels activates calcineurin and dephosphorylates the GABAA receptor complex directly or dephosphorylates proteins critical for the function of the receptor. On the other hand, Ca++ influx activates NO-synthase and induces nitric oxide production, which regulates such receptors via protein kinase G activity.

Only high concentrations of ethanol affect GABAA receptors of rat cerebellum granule cells in culture

In the experiments described in the present report, we evaluated the effects of ethanol on the activity of GABAA receptors of cerebellar granule cells in culture. Only very high ethanol concentrations (100-300 mM) showed a clear and significant stimulatory effect on the activity of such receptors. This result was unexpected. In fact, previous reports from other groups would have suggested high ethanol sensitivity of at least one population of GABAA receptors expressed by granule cells.

Unraveling of important neurobiological mechanisms by the use of pure, fully differentiated neurons obtained from adult animals

An important, and often overlooked, problem in the neurochemical approach to neurobiological problems is that analysis of tissue involves almost always a heterogeneous population of cells (neurons, glia and other types of tissue cells). The use of cell cultures has obvious limitations such as that they derive from embryonic or immediately postnatal animals; in addition, the cell culture conditions most certainly are quite different from the real tissue environment for the nerve cells. We underline here an alternative strategy, which is not new, but which, in our view, has already given formidable contributions to neurobiological studies and still is giving results of great importance. This is the technique proposed and used since the late fifties and early sixties by the senior author (H. Hydén). The method involves the isolation of the big vestibular neurons from the adult rabbit vestibular nucleus. The neurons, fully differentiated and performing a precisely defined function, are obtained rapidly and completely free from surrounding glial cells. The separate microbiochemical study of these cells and their surrounding glia has yielded already in 1962, the information that modifications in gene expression are associated with plastic modifications of the function of the relevant neurons, which take place in the behavioral event of learning. Another important concept was formulated in the same time period following determination of the activities of energy metabolism related enzymes separately in vestibular neurons and their glia under vestibular stimulation. This is the concept that, under increased functional activity glia increases its anaerobic metabolism and passes then on the resulting metabolites to the neurons for aerobic metabolism. Both these concepts (RNA and memory; metabolic cooperation between glia and neurons) are nowadays widely accepted. In addition, this approach with pure big nerve cells has allowed in recent years the discovery of a novel mechanism for chloride extrusion in these cells. This mechanism utilizes structures similar to GABA activated chloride channels in cyclic modifications resulting in the final extrusion of chloride ions. The energy for the process is provided by a protein phosphorylation step. Future approaches are warranted such as the possibility of recognizing by RT-PCR specific neuronal mRNAs and their modification in expression in relation to function and plastic modifications (learning). Another possible interesting application appears to be the recognition of the mRNAs for GABA(A) receptor subunits expressed here in these neurons in relation to the physiological and pharmacological characteristics of these native neuronal GABA(A) receptors.

Binding of paroxetine to the serotonin transporter in membranes from different cells, subcellular fractions and species.

The binding of [3H]-paroxetine to membrane serotonin transporter (SERT) has been studied in membranes from different sources and subcellular fractions. From rat were membranes from venous blood platelets, brain total cortex, brain microsomes, brain crude and purified synaptosomes. Membranes were obtained from venous blood platelets from human volunteers and from brain cortex tissue from neurosurgery (cerebral lobectomies following craniocerebral injuries). The main finding was that the KD of paroxetine binding to the SERT was the same for platelet and nerve ending (synaptosomal) membranes. That parameter was significantly lower in membranes from brain microsomes and cortex total tissue. No species related difference was found, where comparison was possible, between human and rat tissue. The equality of KD of paroxetine binding to blood platelet membranes and to membranes from nerve endings appears to encourage the use of such membranes as a model for brain SERT. Binding at two different temperatures for several of the fractions suggests that paroxetine–SERT interaction is entropy-driven.

New 1,5-benzodiazepine compounds: activity at native GABAA receptors

Various new 1,5-benzodiazepine compounds were synthesized and tested for their biological activity in terms of effects on GABA(A) receptors of rat cerebellar granules in culture. Their effects were compared to those of a 1,4-benzodiazepine agonist, flunitrazepam and the already known 1,5-benzodiazepine antiepileptic clobazam. The effects were evaluated for the two different GABA(A) receptor populations present in these neurons, one mediating phasic inhibition and the other one mediating tonic inhibition. Many such compounds display a profile of inverse agonist to both GABA(A) receptor populations. One of them presents a profile of full agonist at the component mediating phasic inhibition. Interestingly, substitution of just one oxygen atom in that compound with sulphur in a specific position of a morpholine ring resulted in a remarkable change of activity from full agonist to a probable inverse agonist. This indicates such a position as a proton accepting one for the ligand within the benzodiazepine binding pocket of the relevant GABA(A) receptors. In addition, that position appears to be critical for the pharmacological activity.