Janique Guiramand

Anxiolytic properties of green tea polyphenol (-)-epigallocatechin gallate (EGCG).

Naturally occurring polyphenols are potent antioxidants. Some of these compounds are also ligands for the GABA(A) receptor benzodiazepine site. This feature endows them with sedative properties. Here, the anxiolytic activity of the green tea polyphenol (-)-epigallocatechin gallate (EGCG) was investigated after acute administration in mice, using behavioral tests (elevated plus-maze and passive avoidance tests) and by electrophysiology on cultured hippocampal neurons. Patch-clamp experiments revealed that EGCG (1-10 muM) had no effect on GABA currents. However, EGCG reversed GABA(A) receptor negative modulator methyl beta-carboline-3-carboxylate (beta-CCM) inhibition on GABA currents in a concentration dependent manner. This was also observed at the level of synaptic GABA(A) receptors by recording spontaneous inhibitory synaptic transmission. In addition, EGCG consistently inhibited spontaneous excitatory synaptic transmission. Behavioral tests indicated that EGCG exerted both anxiolytic and amnesic effects just like the benzodiazepine drug, chlordiazepoxide. Indeed, EGCG in a dose-dependent manner both increased the time spent in open arms of the plus-maze and decreased the step-down latency in the passive avoidance test. GABA(A) negative modulator beta-CCM antagonized EGCG-induced amnesia. Finally, state-dependent learning was observable after chlordiazepoxide and EGCG administration using a modified passive avoidance procedure. Optimal retention was observed only when animals were trained and tested in the same state (veh-veh or drug-drug) and significant retrieval alteration was observed in different states (veh-drug or drug-veh). Moreover, EGCG and chlordiazepoxide fully generalized in substitution studies, indicating that they induced indistinguishable chemical states for the brain. Therefore, our data support that EGCG can induce anxiolytic activity which could result from an interaction with GABA(A) receptors.

Late N-acetylcysteine treatment prevents the deficits induced in the offspring of dams exposed to an immune stress during gestation.

Prenatal infection is a major stressful experience leading to enhanced susceptibility for mental illnesses in humans. We recently reported in rats, that oxidative stress and glutathione (GSH) shortage occurred in fetal male brain after lipopolysaccharide (LPS) to the dams and that these responses might be involved in the neurodevelopmental deficits observed in adolescent offspring. Furthermore, pretreatment with N‐acetylcysteine (NAC) before LPS avoided both delayed synaptic plasticity and mnesic performance deficits. Since NAC is one of the few medications permitted in pregnant women, this study evaluated the ability of NAC to serve as a protective therapy even after the LPS challenge. Pregnant rats received a single ip injection of E. coli LPS, two days before delivery, and were given NAC in their tap water after the LPS. GSH was evaluated at the time of its expected drop in the hippocampus of male fetuses, whereas long‐term potentiation (LTP) in the CA1 area of the hippocampus and spatial memory in the water‐maze were recorded in 28‐day‐old male offspring. Post‐treatment with NAC, four hours after the LPS challenge fully prevented the drop in the GSH hippocampal content. LTP, as well as spatial learning were completely protected. NAC administration at delivery also partially restored the LTP whereas post‐treatment two days later was inefficient. Another set of dams were supplemented with α‐tocopherol prior to LPS exposure, enhancing the α‐tocopherol levels in fetal hippocampus. This treatment did not prevent the LPS‐induced synaptic plasticity impairment. These results point to fetal hippocampal GSH as a major target of the detrimental effects of in utero LPS challenge. The therapeutic window of NAC extends up to birth, suggesting that this drug might be clinically useful even after an immuno‐inflammatory episode.

A M3 muscarinic receptor coupled to inositol phosphate formation in the rat cochlea

Various neuroactive substances, including excitatory and inhibitory amino acids, biogenic amines and neuropeptides, were tested for their ability to stimulate the inositol phosphate (IPs) cascade in the presence of lithium in the rat cochlea. Among them, only the muscarinic agonists (carbachol and oxotremorine M) were able to stimulate the IPs formation in 12-day-old rat cochleas. The carbachol-elicited IPs formation was inhibited by muscarinic antagonists with the following relative order of potency: atropine greater than 4-DAMP much greater than pirenzepine greater than methoctramine = AF-DX 116. This pharmacological profile suggests that the activation of the M3 muscarinic receptor subtype is responsible for the increase in IPs synthesis in the rat cochlea. However, an interaction with a m5 receptor subtype could not be completely excluded. The unusual link of only one receptor subtype with the phosphoinositide breakdown in the cochlea, as opposed to the usual existence of several receptors coupled to this transduction system in other organs such as the brain, suggest a unique role for muscarinic agonists in the cochlea.

Metabotropic glutamate receptors as drug targets.

L-glutamate (Glu), the main excitatory amino acid neurotransmitter in the mammalian central nervous system, is involved in many physiological functions, including learning and memory, but also in toxic phenomena occurring in numerous degenerative or neurological diseases. These functions mainly result from its interaction with Glu receptors (GluRs). The broad spectrum of roles played by glutamate derived from the large number of membrane receptors, which are currently classified in two main categories, ionotropic (iGluRs) and metabotropic (mGluRs) receptors. The iGluRs are ion channels, permeant to Na(+) (Ca(2+)) while the mGluRs belongs to the superfamily of G-protein coupled receptors (GPCRs). Despite continuous efforts over more than two decades, the use of iGluR agonists or antagonists to improve or inhibit excitatory transmission in pathological states still remains a major challenge, though the discovery and development of recent molecules may prove it worthwhile. This probably results form the vital role of fast excitatory transmission in many fundamental physiological functions. Since the discovery of mGluRs, hope has emerged. Indeed, mGluRs are mainly involved in the regulation of fast excitatory transmission. Consequently, it was logically thought that modulating mGluRs with agonists or antagonists might lead to more subtle regulation of fast excitatory transmission than by directly blocking iGluRs. As a result of intensive investigation, new drugs permitting to discriminate between these receptors have emerged. Moreover, a new class of molecules acting as negative or positive allosteric modulators or mGluRs is now available and appears to be promising. In the following, we will review the classification of mGluRs and the functions in which mGluRs are involved. We will focus on their potential as therapeutic targets for improving numerous physiological functions and for different neurodegenerative and neuropsychiatric disorders, which are related to malfunction of Glu signaling in human beings.

Early, time-dependent disturbances of hippocampal synaptic transmission and plasticity after in utero immune challenge.

BACKGROUND Maternal infection during pregnancy is a recognized risk factor for the occurrence of a broad spectrum of psychiatric and neurologic disorders, including schizophrenia, autism, and cerebral palsy. Prenatal exposure of rats to lipopolysaccharide (LPS) leads to impaired learning and psychotic-like behavior in mature offspring, together with an enduring modification of glutamatergic excitatory synaptic transmission. The question that arises is whether any alterations of excitatory transmission and plasticity occurred at early developmental stages after in utero LPS exposure. METHODS Electrophysiological experiments were carried out on the CA1 area of hippocampal slices from prenatally LPS-exposed male offspring from 4 to 190 days old to study the developmental profiles of long-term depression (LTD) triggered by delivering 900 shocks either single- or paired-pulse (50-msec interval) at 1 Hz and the N-methyl-D-aspartate receptor (NMDAr) contribution to synaptic transmission. RESULTS The age-dependent drop of LTD is accelerated in prenatally LPS-exposed animals, and LTD is transiently converted into a slow-onset long-term potentiation between 16 and 25 days old. This long-term potentiation depends on Group I metabotropic glutamate receptors and protein kinase A activations and is independent of NMDArs. Maternal LPS challenge also leads to a rapid developmental impairment of synaptic NMDArs. This was associated with a concomitant reduced expression of GluN1, without any detectable alteration in the developmental switch of NMDAr GluN2 subunits. CONCLUSIONS Aberrant forms of synaptic plasticity can be detected at early developmental stages after prenatal LPS challenge concomitant with a clear hypo-functioning of the NMDAr in the hippocampus. This might result in later-occurring brain dysfunctions.

Carbachol-induced inositol phosphate formation during rat cochlea development

The age related-intensity developmental pattern of the phosphoinositide breakdown, which leads to the formation of intracellular second messengers, was investigated in rat cochleas by measuring the accumulation of inositol phosphates induced by carbachol in the presence of LiCl. The accumulation of the phosphoinositide metabolites elicited by this muscarinic agonist is very low at post-natal day 1 and particularly large during the period between post-natal days 8 and 14 with a peak around day 12. In the 25-day-old rat cochlea, carbachol induced a 2-fold increase in inositol phosphates (IPs) accumulation, with respect to the basal control level. The apparent affinities of the carbachol-induced IPs responses are 49.6, 31.6 and 36.7 microM in cochleas of 12-, 16- and 25-day-old rats, respectively, thus suggesting that the specific developmental changes are rather due to a modification in the number of muscarinic cholinergic receptors than to alterations of the apparent affinity of carbachol for its receptors. This developmental pattern of carbachol-elicited IPs accumulation reveals a striking time coincidence with both the efferent synaptogenesis at the outer hair cells (OHCs) level and the period of increased sensitivity of OHCs to aminoglycoside toxicity. Phosphoinositide breakdown may, consequently, play a role in the maturation of OHCs and their efferent supply. In addition, the remaining IPs response measured at 25 post-natal days indicates that muscarinic agonist-mediated IPs metabolism also occurs in mature cochlea, and might be involved in the regulation of OHCs motility.

Potentiation of glutamatergic agonist-induced inositol phosphate formation by basic fibroblast growth factor is related to developmental features in hippocampal cultures: neuronal survival and glial cell proliferation.

We investigated the modulation by growth factors of phospholipase C (PLC)‐linked glutamate receptors during in vitro development of hippocampal cultures. In defined medium, glial cells represent between 3 and 14% of total cell number. When we added basic fibroblast growth factor (bFGF) 2 h after plating, we found: (i) a neuroprotection from naturally occurring death for up to 5 days; (ii) a proliferation of glial cells from day 3; and (iii) a potentiation of quisqualate (QA)‐induced inositol phosphate (IP) formation from 1 to 10 days in vitro (DIV) and 1s,3r‐amino‐cyclopentane‐1,3‐dicarboxylate (ACPD) response from 3 to 10 DIV. The antimitotic cytosine‐β,d‐arabinofuranoside (AraC) blocked glial cell proliferation induced by bFGF, but not neuroprotection. Under these conditions, the early potentiation of the QA response (1–3 DIV) was not changed, while the ACPD and late QA response potentiations were prevented (5–10 DIV). Epidermal growth factor was not neuroprotective but it induced both glial cell proliferation and late QA or ACPD potentiation. Surprisingly, the early bFGF‐potentiated QA‐induced IP response was blocked by 6,7‐dinitro‐quinoxaline‐2,3‐dione (DNQX), suggesting the participation of ionotropic (rs)‐α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionate (AMPA)/kainate (KA) receptors. The delayed bFGF‐potentiated ACPD‐induced IP response is inhibited by (s)‐α‐methyl‐4‐carboxyphenylglycine (MCPG), indicating possible activation of glial metabotropic receptors. These results suggest that, in hippocampal cultures, bFGF modulates AMPA and metabotropic glutamate receptors linked to the IP cascade, possibly in relation to the regulation of neuronal survival and glial cell proliferation, respectively.

Potent activation of FGF-2 IRES-dependent mechanism of translation during brain development

Fibroblast growth factor-2 (FGF-2) plays a fundamental role in brain functions. This role may be partly achieved through the control of its expression at the translational level via an internal ribosome entry site (IRES)-dependent mechanism. Transgenic mice expressing a bicistronic mRNA allowed us to study in vivo and ex vivo where this translational mechanism operates. Along brain development, we identified a stringent spatiotemporal regulation of FGF-2 IRES activity showing a peak at post-natal day 7 in most brain regions, which is concomitant with neuronal maturation. At adult age, this activity remained relatively high in forebrain regions. By the enrichment of this activity in forebrain synaptoneurosomes and by the use of primary cultures of cortical neurons or cocultures with astrocytes, we showed that this activity is indeed localized in neurons, is dependent on their maturation, and correlates with endogenous FGF-2 protein expression. In addition, this activity was regulated by astrocyte factors, including FGF-2, and spontaneous electrical activity. Thus, neuronal IRES-driven translation of the FGF-2 mRNA may be involved in synapse formation and maturation.

Excitatory Amino Acid Receptors and Phosphoinositide Breakdown: Facts and Perspectives

Although phosphoinositides were isolated from brain membranes by Folch in 1949, stimulated phosphoinositide metabolism by neuroactive substances was discovered in 1953 by Hokin and Hokin, who showed that the incorporation of 32P into phospholipids of the pancreas was augmented by acetylcholine. In 1961, Dittmer and Dawson (Dawson and Dittmer, 1961; Dittmer and Dawson, 1961) identified these polyphosphoinositides as phosphatidylinositol-4-phosphate (PIP) and phosphatidylinositol-4,5-bisphosphate (PIP2). However, the notion that phosphoinositide metabolism is associated with receptor function was only proposed in 1969 by Durell and co-workers, while a few years later (1975) Michell assumed a close relation between phosphoinositide metabolism and intracellular calcium. Finally, the missing link (inositol trisphosphate) between membrane receptor-coupled phosphoinositide metabolism and the changes in intracellular calcium originating from internal stores was discovered by Berridge and Irvine in 1984.

Astrocytes repress the neuronal expression of GLAST and GLT glutamate transporters in cultured hippocampal neurons from embryonic rats.

Glutamate extracellular levels are regulated by specific transporters. Five subtypes have been identified. The two major ones, GLAST and GLT (glutamate transporters 1 and 2, respectively), are localized in astroglia in normal mature brain. However, in neuron-enriched hippocampal cultures, these proteins are expressed in neurons during the early in vitro development (Plachez et al., 2000). Here, we show that, in these cultures, GLAST and GLT neuronal expression is transient and no longer observed after 7 days in vitro, a stage at which the few astrocytes present in the culture are maturing. Moreover, we demonstrate that these few astrocytes are responsible for the repression of this neuronal expression. Indeed, addition of conditioned medium prepared from primary cultures of hippocampal astrocytes, to cultured hippocampal neurons, rapidly leads to the suppression of neuronal GLAST expression, without affecting neuronal GLT expression. However, when neurons are seeded and co-cultured on a layer of hippocampal astrocytes, they do not develop any immunoreactivity towards GLAST or GLT antibodies. Altogether, these results indicate that glia modulate the expression of GLAST and GLT glutamate transporters in neurons, via at least two distinct mechanisms. Neuronal GLAST expression is likely repressed via the release or the uptake of soluble factors by glia. The repression of neuronal GLT expression probably results from glia-neuron interactions. This further reinforces the fundamental role of direct or indirect neuron-glia interactions in the development of the central nervous system.