Synthesis of -glycoside analogues of isopropyl β-D-1-thiogalactopyranoside (IPTG) and 1-β-D-galactopyranosyl-2-methylpropane. Conformational analysis and evaluation as inhibitors of the lac repressor in and as galactosidase inhibitors.

Isopropyl 1-thio-β-D-galactopyranoside (IPTG, 1) is used widely as an inducer of protein expression in and 1-β-D-galactopyranosyl-2-methylpropane (2), a -glycoside analogue of 1, has also been identified as an inducer. Here, synthesis and study of mimetics of 1 and 2, 1-β-D-galactopyranosyl-2-methylpropan-1-ols and two cyclic acetals derivatives, that constrain the presentation of the iPr group in various geometries is described. Conformational analysis of -glycosides in protic solvent is performed using (i) Desmond metadynamics simulations (OPLS4) and (ii) use of values obtained by H-NMR spectroscopy.

Phosphorylation of glutamate receptor interacting protein 1 regulates surface expression of glutamate receptors.

The number of synaptic alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)-type glutamate receptors (AMPARs) controls the strength of excitatory transmission. AMPARs cycle between internal endosomal compartments and the plasma membrane. Interactions between the AMPAR subunit GluR2, glutamate receptor interacting protein 1 (GRIP1), and the endosomal protein NEEP21 are essential for correct GluR2 recycling.

Central nervous system functions of PAK protein family: from spine morphogenesis to mental retardation.

Several of the genes currently known to be associated, when mutated, with mental retardation, code for molecules directly involved in Rho guanosine triphosphatase (GTPase) signaling. These include PAK3, a member of the PAK protein kinase family, which are important effectors of small GTPases. In many systems, PAK kinases play crucial roles regulating complex mechanisms such as cell migration, differentiation, or survival.

Long-term rearrangements of hippocampal mossy fiber terminal connectivity in the adult regulated by experience.

We investigated rearrangements of connectivity between hippocampal mossy fibers and CA3 pyramidal neurons. We found that mossy fibers establish 10-15 local terminal arborization complexes (LMT-Cs) in CA3, which exhibit major differences in size and divergence in adult mice. LMT-Cs exhibited two types of long-term rearrangements in connectivity in the adult: progressive expansion of LMT-C subsets along individual dendrites throughout life, and pronounced increases in LMT-C complexities in response to an enriched environment.

Glutamate receptor changes associated with transient anoxia/hypoglycaemia in hippocampal slice cultures.

Transient anoxia/hypoglycaemia in organotypic hippocampal slice cultures, a model of transient brain ischaemia, ultimately results in delayed cell death. Although the mechanisms underlying this delayed death remain unknown, an increase in excitatory drive has been postulated. We report here that transient anoxia/hypoglycaemia in rat hippocampal slice cultures resulted in a 70-80% enhancement of evoked, alpha-amino-3-hydroxy-5-methyl-4-isoxazolpropionic acid (AMPA) receptor-mediated, excitatory responses lasting over 60 min.

Application of photoconversion technique for correlated confocal and ultrastructural studies in organotypic slice cultures.

Photoconversion of fluorescent staining into stable diaminobenzidine (DAB) precipitate is widely used for neuroanatomical and developmental studies. An important advantage of the approach is to make correlations between light and electron microscopy analyses possible, the DAB reaction product formed during photoconversion being electron dense. By combining a photoconversion approach with biolistic transfection of neurons in organotypic hippocampal slice cultures, we describe here a methodology that allowed us to study at the electron microscopy level the fine details of cells expressing specific genes of interest.

The endosomal protein NEEP21 regulates AMPA receptor-mediated synaptic transmission and plasticity in the hippocampus.

The neuron-enriched endosomal protein 21 (NEEP21) has recently been implicated in the regulation of AMPA receptor (AMPAR) trafficking and proposed to participate in the control of synaptic strength. We tested here this possibility at CA3-CA1 synapses in hippocampal slice cultures using antisense-mediated down-regulation of NEEP21 expression or transfection of a fragment of the cytosolic domain of NEEP21. We found that NEEP21 suppression or expression of the dominant-negative fragment reduced spontaneous and evoked AMPAR-mediated synaptic currents without affecting presynaptic properties.

The mental retardation protein PAK3 contributes to synapse formation and plasticity in hippocampus.

Mutations of the gene coding for PAK3 (p21-activated kinase 3) are associated with X-linked, nonsyndromic forms of mental retardation (MRX) in which the only distinctive clinical feature is the cognitive deficit. The mechanisms through which PAK3 mutation produces the mental handicap remain unclear, although an involvement in the mechanisms that regulate the formation or plasticity of synaptic networks has been proposed. Here we show, using a transient transfection approach, that antisense and small interfering RNA-mediated suppression of PAK3 or expression of a dominant-negative PAK3 carrying the human MRX30 mutation in rat hippocampal organotypic slice cultures results in the formation of abnormally elongated dendritic spines and filopodia-like protrusions and a decrease in mature spine synapses.

Activity-induced changes of spine morphology.

Spine morphology has been shown in recent years to exhibit a high degree of plasticity. In developing tissue such as organotypic slice cultures, shape changes in spines as well as reorganization of the postsynaptic density (PSD) occur within minutes. Furthermore, several studies have shown that these and other changes can be induced by or are dependent on synaptic activation.

LTP, memory and structural plasticity.

Our current understanding of the mechanisms of information processing and storage in the brain, based on the concept proposed more than fifty years ago by D. Hebb, is that a key role is played by changes in synaptic efficacy induced by coincident pre- and postsynaptic activity. Decades of studies of the properties of long-term potentiation (LTP) have shown that this form of plasticity adequately fulfills these requirements and is likely to contribute to several models of learning and memory.

A point mutant of GAP-43 induces enhanced short-term and long-term hippocampal plasticity.

The growth-associated protein GAP-43 (or neuromodulin or B-50) plays a critical role during development in mechanisms of axonal growth and formation of synaptic networks. At later times, GAP-43 has also been implicated in the regulation of synaptic transmission and properties of plasticity such as long-term potentiation. In a molecular approach, we have analyzed transgenic mice overexpressing different mutated forms of GAP-43 or deficient in GAP-43 to investigate the role of the molecule in short-term and long-term plasticity.

Remodeling of hippocampal synaptic networks by a brief anoxia-hypoglycemia.

Cerebral ischemia is a major cause of brain dysfunction. Using a model of delayed death induced by a brief, transient oxygen and glucose deprivation, we studied here how this affected the structural organization of hippocampal synaptic networks. We report that brief anoxic-hypoglycemic episodes rapidly modified the structure of synapses.

Involvement of calmodulin-dependent protein kinase II in carbachol-induced rhythmic activity in the hippocampus of the rat.

The role of calcium and protein kinases in rhythmic activity induced by muscarinic receptor activation in the CA1 area in rat hippocampal slices was investigated. Extracellular recording showed that carbachol (20 microM) induced synchronized field potential activity with a dominant frequency of 7.39+/-0.

Vasopressin and oxytocin action in the brain: cellular neurophysiological studies.

During the last two decades it has become apparent that vasopressin (VP) and oxytocin (OT), in addition to playing a role as peptide hormones, also act as neurotransmitters. Morphological studies and electrophysiological recordings have shown a close anatomical correlation between the presence of these receptors and the neuronal responsiveness to VP or OT. These compounds have been found to affect membrane excitability in neurons located in the hippocampus, hypothalamus, lateral septum, brainstem, spinal cord and superior cervical ganglion.

The oxytocin-induced inward current in vagal neurons of the rat is mediated by G protein activation but not by an increase in the intracellular calcium concentration.

The neuropeptide oxytocin can depolarize parasympathetic preganglionic neurons in the dorsal motor nucleus of the vagus nerve of the rat by generating a sustained inward current, which is sodium-dependent and tetrodotoxin-insensitive. The second messenger activated by oxytocin receptor binding is, however, not yet known. In the present study, we attempted to characterize it by using the whole-cell recording technique and brainstem slices.

Action of a metabotropic glutamate receptor agonist in rat lateral septum: induction of a sodium-dependent inward aftercurrent.

The mechanism by which (1S,3R)-ACPD, a metabotropic glutamate receptor agonist, induces burst firing in lateral septal neurons of the rat was investigated in coronal brainstem slices. Membrane currents were characterized in voltage clamp using whole-cell recordings. In the presence of (1S,3R)-ACPD, following depolarizing voltage jumps, repolarization towards the holding potential generated an inward aftercurrent.

Whole-cell NMDA-evoked current in suprachiasmatic neurones of the rat: modulation by extracellular calcium ions.

The action of N-methyl-D-aspartic acid (NMDA) on suprachiasmatic neurones was studied using whole-cell recordings in coronal hypothalamic slices of the rat. The location of the recorded neurones within the suprachiasmatic nucleus was ascertained by intracellular labelling with biocytin, followed by histological processing of the slice. Suprachiasmatic neurones had an input resistance of 780 +/- 20 M omega (mean +/- S.

Axotomized neonatal motoneurons overexpressing the bcl2 proto-oncogene retain functional electrophysiological properties.

Bcl2 overexpression prevents axotomy-induced neuronal death of neonatal facial motoneurons, as defined by morphological criteria. However, the functional properties of these surviving lesioned transgenic neurons are unknown. Using transgenic mice overexpressing the protein Bcl2, we have investigated the bioelectrical properties of transgenic facial motoneurons from 7 to 20 days after neonatal unilateral axotomy using brain-stem slices and whole cell patch-clamp recording.

Modulation by divalent cations of the current generated by vasopressin in facial motoneurons.

Vasopressin generates a voltage-gated, sodium-dependent current in facial motoneurons in brainstem slices. Reducing the extracellular calcium concentration from 2 to 0.01 mM caused a 30 to 120% increase in the amplitude of this current.