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Alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors are the major excitatory neurotransmitter receptors of the central nervous system. AMPA receptor complexes that contain the AMPA-type glutamate receptor subunit 2 (GluR2) are responsible for the low calcium permeability typical of most AMPA receptors, and the absence of GluR2 may be a key factor in neurotoxicity. A polyclonal antibody was produced to a 16 amino acid peptide near the C-terminus of GluR2 and was affinity-purified in a three-step procedure. The antibody did not recognize other AMPA subunits in transfected cells with the use of either Western blots or immunocytochemistry. This highly specific GluR2 antibody was used to provide a specific morphological study of GluR2 protein distribution in neurons and synapses of the rat. GluR2 is prevalent in most principal neurons throughout the telencephalon. Neurons with few or no GluR2 subunits include two major types: 1) some populations of interneurons of the telencephalon and of some other areas and 2) many populations of principal neurons in the brainstem and spinal cord. Immunofluorescence showed that GluR2 immunolabeling was widespread, including in dendrites and puncta, in the hippocampus and neocortex. Where they were present, GluR2 subunits colocalized with other AMPA receptor subunits in individual neurons. Electron microscopy of the hippocampus showed GluR2-bearing, calcium-impermeable AMPA receptors postsynaptic to dendrite synapses of forebrain principal neurons. In addition, electron microscopy of the neocortex showed significant staining in postsynaptic profiles. Electron microscopy of the cerebellum revealed the presence of GluR2 subunits in the postsynaptic profiles of many parallel fiber/Purkinje cell spine synapses, whereas electron microscopy of the spinal cord showed substantial staining in the postsynaptic profiles of dorsal horn synapses, but not in ventral horn synapses. Both ultrastructural and immunofluorescence data showed that calcium-impermeable AMPA receptors are widespread in dendrite arborizations.

An antibody was made to a C-terminus peptide of the glutamate receptor delta 2 subunit and used to study the distribution, biochemical properties, and developmental expression of the delta receptor in rat brain. The antibody recognizes both delta 1 and delta 2 but not AMPA, kainate, NMDA, and mGluR1 alpha glutamate receptor subunits based on Western blot analysis of transfected HEK-293 cells. Western blot analysis of brain showed a single immunoreactive band, migrating at M(r) = 114,000. Immunoprecipitation of detergent-solubilized cerebellar membranes was done to determine if delta is associated with other glutamate receptor subunits and if it binds any of the common excitatory amino acid ligands. Based on results of these studies, AMPA, kainate, NMDA, and mGluR1 alpha subunits do not coassemble with delta subunits, and 3H-glutamate, 3H-AMPA and 3H-kainate do not bind to the delta receptor complex. Western blot and immunocytochemical analyses showed marked expression of delta in the cerebellum while lower levels were detected in other regions of the brain. A dramatic increase of delta 1/2 immunoreactivity was observed in the cerebellum between the ages of 10 and 15 d postnatal. Light and electron microscopy, respectively, demonstrated dense immunostaining in Purkinje cells and in postsynaptic densities of the adult parallel fiber-Purkinje spine synapse. The prominent delta 1/2 immunoreactivity found in the parallel fiber-Purkinje spine synapse, and the temporal correlation of the development of this synapse with the major increase in delta 1/2 immunoreactivity, suggest a major functional role for the delta subunits in cerebellar circuitry.

International audience; We previously reported that the inositol phosphates (IPs) synthesis is induced by muscarinic agonists in the rat cochlea and that this stimulation is maximal at postnatal day 12. This peak response is concomitant with the onset of the efferent synaptogenesis at the outer hair cell level. Whether the correlation between this neuronal plasticity and the enhanced IPs formation is unique to the rat or a general feature of the developing vertebrate cochlea is not known. To examine this question, we measured, in the presence of LiCI, the accumulation of (3H)-IPs induced by carbachol, in the developing chick cochlear duct during a period ranging from embryonic day (E) 8 to post-hatching day (P) 20. Carbachol (1 mM) causes a significant increase of IPs formation relative to basal values at all ages. This IPs accumulation is maximal at E8 (1854% of the basal level), then, rapidly decreases until P13 when it reaches a steady-state level of 294% of the basal level. Strikingly, this gradual decline in IPs formation is interrupted between El5 and El9, by a transient increase in IPs synthesis. This rise peaks at El6 with a stimulation value of 757% of the control level. This maximal stimulation is inhibited by atropine in a dose-dependent manner, as is the case at E9, suggesting the involvement of muscarinic receptors. Interestingly, the occurrence of the peak response is concomitant with the plastic events associated with the maturation of the efferent innervation of the cochlear duct. Thus, these results suggest that there may be a correlation between cochlear plasticity and enhanced IPs synthesis, which is not species-specific. The possible significance of the overall decrease in IPs formation, occurring during embryonic development , is discussed. The degradation of membrane phosphatidylinositol 4,5 biphosphate, by the enzyme phospholi-pase C, leads to the formation of diacylglycerol and inositol phosphates (IPs). Among these metabolites, diacylglycerol and inositol 1,4,5-trisphosphate are considered as second messengers. 4 The former directly activates the protein kinase C enzymes; the latter elicits a massive release of calcium from intracellular stores. This transduction system has been found to be driven by specific agonist-activated receptors such as muscarinic cholinergic receptors. 7 We previously reported that this transduction system is stimulated, in the rat cochlea, by muscarinic cholinergic agonists probably, via the activation of a M3 muscarinic receptor, s During the postnatal development of the mammalian cochlea, the muscarinic agonist-induced IPs formation is characterized by a peak around postnatal day 12. 3 This peak coincides with a time period during which plastic events lead to the setting up of the mature efferent innervation of the outer hair cells of the organ of Corti. I1 These efferent terminals are thought to be cholinergic. 6 Thus, it is conceivable that the IPs metabolism may play a role in cochlear neural plasticity. Whether this concomitance, between the increased IPs synthesis and the efferent synaptogenesis, is an overall developmental process in the vertebrate inner ear or is a specific feature of the rat cochlea remains to be investigated. To address this question, we studied the pattern of the phosphoinositide breakdown during the development of the chick basiler papilla. Although phylogenetically remote, the avian basilar papilla and the mammalian organ of Corti share some morphological homologies. They both possess, for instance, two types of hair cells lying on a basilar membrane and covered by a tector-ial membrane. The sensory hair cells are innervated, in both classes, by four different types of fibres, two of them belonging to the efferent systems and the other two to the efferent systems. II'16'23'25 Finally, physiological evidence, supporting the possibility of the presence of muscarinic receptors in the chick auditory organ, as this is the case in the rat cochlea, 3,8 is

The change in the conformation of Bax at the onset of apoptosis is a determinant for the execution of this cell death programme. However, very few models can account for this modification and the factors involved in this process remain elusive. We have analysed the modifications in the conformation induced by a variation in pH using a cell-free assay. We show that a moderate basic or acidic pH can induce apoptotic-like changes in the conformation of Bax, such as the exposure of the N-terminal or the BH3 domain. These changes in the conformation are associated with the binding of Bax to mitochondria and an enhanced Bax homo- and oligomerisation. Our results suggest that variations in the pH, in a range consistent with that often observed during apoptosis, are sufficient to trigger Bax translocation to mitochondria and the subsequent release of apoptogenic factors from this organelle.

We investigated the role of cholinesterases in the differentiation of chicken retina and brainstem neurons in vitro, using antisense oligonucleotides. When cultures were treated with antisense oligonucleotides which are complementary to the region of the initiation codon or to the junction of consecutive exons of the chicken acetylcholinesterase (AChE) gene, we observed a complete retraction of all neuntes: the cells became spherical and formed compact globular aggregates. These morphological changes were only accompanied by a minor decrease in AChE enzymatic activity. They were already visible after 8 hours following administration of antisense oligonucleotides and lasted for 2 to 3 days. At that point, some cells started to extend neuntes again, and this process could be blocked again by a second addition of antisense oligonucleotides so that the aggregated state could be maintained up to two weeks in culture. Therefore, the effect of oligonucleotides was not due to toxicity. Its specificity was shown by the fact that sense oligonucleotides or scrambled oligonucleotides had no effect. Moreover antisense oligonucleotides, directed against butyrylcholinesterase (BChE) did not induce any morphological change. These results strongly suggest that AChE may be involved in neuronal differentiation via structural interactions, in addition to its catalytic activity.

This chapter is based on the Bert La Du — ASPET plenary lecture delivered by Jean Massoulie at the opening of the Sixth International Meeting on Cholinesterases. It is a pleasure and an honour to dedicate this discussion of the diversity of acetylcholinesterase forms to Bert La Du, whose in depth approach to the molecular pharmacogenetics of the sister enzyme, butyrylcholinesterase, has always been a source of inspiration for us, because of the strong similarities and considerable differences between the two enzyme systems.

Neuronal loss and gliosis were detected in the rat hippocampus soon after unilateral intra-amygdala injection of kainate (KA) (2.5 nmol) while solid mossy fiber sprouting could be seen only fourteen days after this injection. Using this experimental model, we examined the metabotropic glutamate receptor (mGluR)-induced inositol phosphate (IP) formation in hippocampal synaptoneurosomes and slices. In synaptoneurosomes prepared from ipsilateral hippocampi fourteen days following injection, there were no significant changes in mGluR- and carbachol(CARB)-stimulated IPs syntheses when sham-operated and KA-injected animals were compared. In the corresponding hippocampal slices, significant increases of the mGluR responses mediated by ibotenate (IBO) and aminocyclopentane-trans-1,3-dicarboxylate (t-ACPD) were noted after KA application. The net stimulation values respectively expressed in a pair-wise fashion for buffer-injected control and KA-treated animals were IBO: 1,947 +/- 457 and 10,553 +/- 1,242; t-ACPD: 1,557 +/- 662 and 9,449 +/- 2,251 dpm/mg protein respectively. Significantly augmented mGluR responses in hippocampal slices were also measured at 7, 42 and 92 days after KA injection. There were, however, no significant increases in CARB-stimulated phosphoinositide hydrolysis in the hippocampal slices at all time-intervals after KA administration. These findings show that there are differences between the mGluR responses in hippocampal synaptoneurosome and slice preparations, suggesting the presence of two distinct populations of mGluR in each of these two models. The large specific increases in certain mGluR activities after KA-induced status epilepticus in hippocampal slices could represent one of the molecular mechanisms which underlie the profound morphological changes, in particular gliosis or mossy fiber sprouting, which follow the KA-induced status epilepticus.

The effect of postnatal age on phosphoinositide metabolism per se and on quisqualate-stimulated phosphoinositide metabolism was characterized in synaptoneurosomes prepared from nine different regions of the rat nervous system, namely the brainstem, cerebellum, cerebral cortex, colliculi, hippocampus, hypothalamus, olfactory bulb, spinal cord and striatum. In the hippocampus, striatum, cerebellum, cerebral cortex, brainstem, colliculus and spinal cord, the basal levels of inositol phosphate (inositol-1-phosphate+inositol-4,5-bisphosphate) formation were maximal two days after birth and declined steeply to steady-state levels from the age of 10 postnatal days. Similarly, in the olfactory bulb, basal inositol phosphate synthesis did not significantly change when measured during the period from postnatal day 10 to 42. The extent of [ 3 H]-inositol labelling of phosphoinositides as a function of age presented similar profiles when measured in hippocampal, striatal, cerebellar and cerebral cortical synaptoneurosomes, i.e. maximal at perinatal ages and minimal at adult ages. In the hypothalamus, [ 3 H]-inositol labelling of phosphoinositides showed an increase from postnatal day 12 to higher levels from postnatal days 14 to 18 subsequently followed by a dramatic increase from postnatal day 21 to 42. A similar developmental trend was also obtained for basal inositol phosphate synthesis. On the whole, four types of developmental profiles for quisqualate-stimulated inositol phosphate formation (expressed as the percentage of the basal level and as the difference between stimulated and basal levels of radioactive inositol phosphates) were obtained depending on the nervous system region studied. In the early, prenatally developed nervous system regions, namely the brainstem and the spinal cord, no postnatal stimulation peaks of quisqualate-induced inositol phosphate formation were recorded. This was also the case for the colliculi when the stimulation of IP formation was expressed as the difference in basal and stimulated levels of inositol phosphates. Secondly, in the olfactory bulb a region known to possess a continuous capacity for developmental plasticity both structurally and functionally during the first three weeks of postnatal development, a simultaneous sustained high level of quisqualate stimulation of phosphoinositide metabolism (fluctuating around 200% of the basal level) during the early postnatal period was evident. Thirdly, in regions of the central nervous system like the cerebellum, cerebral cortex, hippocampus and the striatum known to undergo intense developmental activity during the first two postnatal weeks, peaks of quisqualate-stimulated phosphoinositide metabolism were initially detected around the first week after birth in each of these brain areas. Finally, in the hypothalamus where structurally unique postnatal developmental processes are known to take place, quisqualate-induced inositol phosphate formation progressively increases with age to reach maxima at postnatal day 18. The transient increases in quisqualate responses in the cerebellum, hippocampus and striatum are probably specific to quisqualate since carbachol-stimulated phosphoinositide metabolism yielded different age-associated response patterns. Similar increases of carbachol- and quisqualate-mediated phosphoinositide hydrolysis were on the other hand assayed in cerebral cortical and hypothalamic synaptoneurosomes. EC 50 , values for quisqualate (the quisqualate concentration required to produce 50% of the maximal effect) at postnatal days 6 and 10 were not significantly different in each of four types of synaptoneurosomes: cerebellar, cerebral cortical, hippocampal and striatal. On the basis of these latter results, it was deduced that the peak of quisqualate-stimulated phosphoinositide metabolism does not materialize on the basis of changes in quisqualate metabotropic receptor affinity. In conclusion, the measurement of inositol phosphate formation in synaptoneurosomes prepared from different regions of the postnatally developing nervous system indicate that there is a temporal correlation between the increased activity of quisqualate-stimulated phosphoinositide metabolism mediated by specific metabotropic glutamate receptors and region-specific developmental events. This could suggest a key role for certain metabotropic glutamate receptors in the developmental plasticity of the nervous system.

The characteristics of the transduction mechanism(s) activated by glutamate (Glu) via the quisqualate metabotropic receptor, as well as by depolarizing agents, to trigger formation of inositol phosphates (IPs) were investigated in 8-day-old rat forebrain synaptoneurosomes. The replacement of external Na+ by various compounds (Li+, Tris+, N-methyl-D-glucamine+, and sucrose) induces an increase in basal accumulation of IPs and depolarizes synaptoneurosome membranes. Under these conditions, Glu- and K(+)-induced accumulations of IPs are inhibited, whereas the carbachol (Carb)-elicited response of IPs parallels the basal one. Agents increasing Na+ influx, such as veratridine and monensin, depolarize synaptoneurosomes and stimulate formation of IPs. These stimulations are not additive with responses of IPs elicited by Glu or K+. These data suggest that (a) Glu activates phosphoinositide metabolism via a specific mechanism (distinct from that of cholinergic agonists), (b) depolarizing agents and Glu share at least one common intermediate step in their mechanisms of activation of the metabolism of IPs, and (c) the depolarization may correspond to this common step. In addition, Na+ seems to be required for Glu stimulation of metabolism of IPs. The depolarization associated with the action of Glu on formation of IPs results neither from an influx via tetrodotoxin-sensitive voltage-dependent Na+ channels nor from an entry via the classically characterized Na+/Ca2+ or Na+/H+ exchangers. In fact, tetrodotoxin (2 microM) has no effect on the Glu- or K(+)-elicited response of IPs. Amiloride (greater than 50 microM) and some of its derivatives similarly inhibit not only Glu- and K(+)- but also Carb-evoked formation of IPs.

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.

International audience; 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.

Oligonucleotides induce various cellular responses which are not due to the blockade of protein synthesis by an antisense mechanism. Oligonucleotides presenting double-stranded or G-quartet structures (ribo- or deoxyribonucleotides, phosphodiester or phosphorothioated) induce retraction of neurites and aggregation of chicken retinal cells within 10–20 h. This effect is reversible, non-toxic; it appears to require internalization and can be mimicked by treatment of the cells with an RGDS peptide. The oligonucleotides appear to trigger a cascade of intracellular events, affecting the adhesive properties of integrins. In addition, a subset of oligonucleotides induced platelet aggregation, probably through their interaction with membrane receptors. Recognition of these effects is important for the design and interpretation of antisense experiments.