Barrel cortex development lacks a key stage of hyperconnectivity from deep to superficial layers in a rat model of Absence Epilepsy.

During development of the sensory cortex, the ascending innervation from deep to upper layers provides a temporary scaffold for the construction of other circuits that remain at adulthood. Whether an alteration in this sequence leads to brain dysfunction in neuro-developmental diseases remains unknown. Using functional approaches in a genetic model of Absence Epilepsy (GAERS), we investigated in barrel cortex, the site of seizure initiation, the maturation of excitatory and inhibitory innervations onto layer 2/3 pyramidal neurons and cell organization into neuronal assemblies. We found that cortical development in GAERS lacks the early surge of connections originating from deep layers observed at the end of the second postnatal week in normal rats and the concomitant structuring into multiple assemblies. Later on, at seizure onset (1 month old), excitatory neurons are hyper-excitable in GAERS when compared to Wistar rats. These findings suggest that early defects in the development of connectivity could promote this typical epileptic feature and/or its comorbidities.

Novel N-aryl nicotinamide derivatives: Taking stock on 3,6-diazabicyclo[3.1.1]heptanes as ligands for neuronal acetylcholine receptors.

We designed the synthesis of a small library of 3-substituted-3,6-diazabicyclo[3.1.1]heptanes whose affinity on neuronal nicotinic receptors (nAChRs) was evaluated. Among the synthesized compounds, the 5-(3,6-diazabicyclo[3.1.1]heptane-3-yl)-N-(2-fluorophenyl)nicotinamide 43 proved to be the most interesting compound with α4ß2Ki value of 10 pM and a very high α7/α4ß2 selectivity. Furthermore, compounds 35, 39 and 43 elicited a selective partial agonist activity for α4ß2 nAChR subtype. Finally, in this paper we also report the conclusions on the 3,6-diazabicyclo[3.1.1]heptanes as ligands for nAChRs, resulting from our consolidated structure activity relationship (SAR) studies on this template.

Extracellular mild acidosis decreases the Ca2+ permeability of the human NMDA receptors.

NMDA receptors (NMDARs) are glutamate-gated ion channels involved in excitatory synaptic transmission and in others physiological processes such as synaptic plasticity and development. The overload of Ca2+ ions through NMDARs, caused by an excessive activation of receptors, leads to excitotoxic neuronal cell death. For this reason, the reduction of Ca2+ flux through NMDARs has been a central focus in finding therapeutic strategies to prevent neuronal cell damage. Extracellular H+ are allosteric modulators of NMDARs. Starting from previous studies showing that extracellular mild acidosis reduces NMDA-evoked whole cell currents, we analyzed the effects of this condition on the NMDARs Ca2+ permeability, measured as "fractional calcium current" (Pf, i.e. the percentage of the total current carried by Ca2+ ions), of human NMDARs NR1/NR2A and NR1/NR2B transiently transfected in HeLa cells. Extracellular mild acidosis significantly reduces Pf of both human NR1/NR2A and NR1/NR2B NMDARs, also decreasing single channel conductance in outside out patches for NR1/NR2A receptor. Reduction of Ca2+ flux through NMDARs was also confirmed in cortical neurons in culture. A comparative analysis of both NMDA evoked Ca2+ transients and whole cell currents showed that extracellular H+ differentially modulate the permeation of Na+ and Ca2+ through NMDARs. Our data highlight the synergy of two distinct neuroprotective mechanisms during acidosis: Ca2+ entry through NMDARs is lowered due to the modulation of both open probability and Ca2+ permeability. Furthermore, this study provides the proof of concept that it is possible to reduce Ca2+ overload in neurons modulating the NMDAR Ca2+ permeability.

Pyridinyl- and pyridazinyl-3,6-diazabicyclo[3.1.1]heptane-anilines: Novel selective ligands with subnanomolar affinity for α4ß2 nACh receptors.

The cholinergic pathways in the central nervous system (CNS) of animals and humans are important for cognitive and behavioural functions. Until a few years ago, it was thought that the key molecules transducing the cholinergic message were the metabotropic muscarinic receptors, but it is now known that ionotropic neuronal nicotinic receptors (nAChRs) are also involved. Based on recent studies, we prepared a small library of novel 3-substituted-3,6-diazabicyclo [3.1.1]heptanes, whose binding activity and functionality have been assayed. Among the synthesized compounds, the 3-(anilino)pyridine series resulted in the most interesting compounds with α4ß2Ki values ranging from 0.0225 nM (12g) to 2.06 nM (12o).

The fifth subunit in α3ß4 nicotinic receptor is more than an accessory subunit.

The α3ß4 subtype is the predominant neuronal nicotinic acetylcholine receptor present in the sensory and autonomic ganglia and in a subpopulation of brain neurons. This subtype can form pentameric receptors with either 2 or 3 ß4 subunits that have different pharmacologic and functional properties. To further investigate the role of the fifth subunit, we coexpressed a dimeric construct coding for a single polypeptide containing the ß4 and α3 subunit sequences, with different monomeric subunits. With this strategy, which allowed the formation of single populations of receptors with unique stoichiometry, we demonstrated with immunofluorescence and biochemical and functional assays that only the receptors with 3 ß4 subunits are efficiently expressed at the plasma membrane. Moreover, the LFM export motif of ß4 subunit in the fifth position exerts a unique function in the regulation of the intracellular trafficking of the receptors, their exposure at the cell surface, and consequently, their function, whereas the same export motif present in the ß4 subunits forming the acetylcholine binding site is dispensable.-Crespi, A., Plutino, S., Sciaccaluga, M., Righi, M., Borgese, N., Fucile, S., Gotti, C., Colombo, S. F. The fifth subunit in α3ß4 nicotinic receptor is more than an accessory subunit.

From pyrrolidinyl-benzodioxane to pyrrolidinyl-pyridodioxanes, or from unselective antagonism to selective partial agonism at α4ß2 nicotinic acetylcholine receptor.

Each of the four aromatic -CH= of (S,R)-2-pyrrolidinyl-1,4-benzodioxane [(S,R)-6] and of its epimer at the dioxane stereocenter (S,S)-6, previously reported as α4ß2 nAChR ligands, was replaced with nitrogen. The resulting four diastereoisomeric pairs of pyrrolidinyl-pyridodioxanes were studied for the nicotinic affinity and activity at α4ß2, α3ß4 and α7 nAChR subtypes and compared to their common carbaisostere. It turned out that such isosteric substitutions are highly detrimental, but with the important exception of the S,R stereoisomer of the pyrrolidinyl-pyridodioxane with the pyridine nitrogen adjacent to the dioxane and seven atoms distant from the pyrrolidine nitrogen. Indeed, this stereo/regioisomer not only maintained the α4ß2 affinity of [(S,R)-6], but also greatly improved in selectivity over the α3ß4 and α7 subtypes and, most importantly, exhibited a highly selective α4ß2 partial agonism. The finding that [(S,R)-6] is, instead, an unselective α4ß2 antagonist indicates that the benzodioxane substructure confers affinity for the α4ß2 nAChR binding site, but activation of this receptor subtype needs benzodioxane functionalization under strict steric requirements, such as the previously reported 7-OH substitution or the present isosteric modification.