Characterizing the hippocampal theta's response to carbachol; using a complete septo-hippocampal preparation / Caracterización de la respuesta theta del hipocampo al carbacol en una preparación septo-hipocámpica completa

Abstract: The present study describes the pharmacological analysis of the effects of carbachol, a cholinergic agonist, on hippocampal theta activity. Knowing that this activity is critically related to cognitive function and altered in patients with neurodegeneration, pharmacological efforts aiming to directly modulate hippocampal theta activity becomes of central importance. In a recently developed complete septo-hippocampal preparation, carbachol elicited significant theta power enhancement with 1 μM. Concentrations under 1 μM and over 2 μM carbachol caused significant reduction in the power of hippocampal theta activity. Carbachol effects were completely blocked with the cholinergic antagonist scopolamine. At the experimental level, it is the first time the direct action of a cholinergic agonist is evaluated in the septo-hippocampal pathway completely isolated. However, carbachol as a cholinergic agonist is a drug with a certain level of nonspecific response. That is why to correct this experimental limitation, we used scopolamine (cholinergic antagonist) which allowed us to corroborate the effects on the cholinergic pathway. In summary, electrophysiological assays demonstrated an effective concentration range of carbachol specifically modulating hippocampal theta activity.

Resumen: El presente estudio describe el análisis farmacológico de los efectos de carbacol, un agonista colinérgico, sobre la actividad theta del hipocampo. Sabiendo que esta actividad está críticamente relacionada con la función cognitiva y alterada en pacientes con neurodegeneración, los esfuerzos farmacológicos destinados a modular directamente la actividad theta del hipocampo se vuelven de gran importancia. En una preparación completa que contiene la región septal media conectada al hipocampo, desarrollada recientemente, 1 μM de carbacol provocó un incremento significativo a nivel de potencia en la actividad theta del hipocampo. Las concentraciones menores de1 μM y mayores a 2 μM causaron una reducción significativa en la potencia de la actividad theta. Los efectos del carbacol fueron completamente bloqueados con la escopolamina, antagonista colinérgico. A nivel experimental, es la primera vez que se evalúa la acción directa de un agonista colinérgico en la vía septo-hipocámpica completamente aislada. Sin embargo, el carbacol como agonista colinérgico es un fármaco que presenta cierto nivel de respuesta inespecífica. Es por eso que para corregir esta limitante experimental, se utilizó escopolamina (antagonista colinérgico) lo que nos permitió corroborar los efectos sobre la vía colinérgica. En resumen, nuestros estudios electrofisiológicos demostraron un intervalo de concentración eficaz del carbacol que modula específicamente la actividad theta del hipocampo.

Phosphorylation of tau protein at sites Ser(396-404) is one of the earliest events in Alzheimer's disease and Down syndrome.

AIMS: Phosphorylation, conformational changes and cleavage of tau protein have been widely suggested to contribute to abnormal tau processing in the pathogenesis of Alzheimer's disease, as well as in other tauopathies. Consistently, many phosphorylated sites, such as Ser(199-202) -Thr(205) and Ser(396-404) , have been associated with this pathological processing. The present study examined the chronological appearance of phosphorylation during the neurofibrillary tangle (NFT) evolution in Alzheimer disease (AD) and Down syndrome. METHODS: Immunohistochemistry for modified tau [phosphorylated at Ser(199-202) -Thr(205) (AT8) and Ser(396-404) (PHF-1) or truncated at D(421) (TauC3) and E(391) (MN423)] was performed on paraffin-embedded human brain sections. Double immunofluorescence for phosphorylated and truncated tau was used to detect intensity and distribution of tau immunoreactivity, and provided detailed characterization of NFT pathology. RESULTS: Phosphorylation at sites Ser(396-404) was significantly increased when compared with phosphorylations at sites Ser(199-202) -Thr(205) . Around 50% of the total structures containing phosphorylation at sites Ser(396-404) were found as early phospho-tau aggregates with a well-preserved neuronal soma. Phosphorylation of tau protein at sites Ser(396) coexists with early and late truncation events. Tau abnormal processing in Down syndrome consistently showed similar alterations as observed in AD. CONCLUSION: Phosphorylation of tau protein at the carboxyl terminus may be among the earliest tau events, and it occurs prior to the apparition of the classical fibrillar structure. Finally, these data validate PHF-1 as an efficient marker for AD cytopathology following the progression of tau aggregation into NFT.

Conformational changes and cleavage of tau in Pick bodies parallel the early processing of tau found in Alzheimer pathology.

Neuronal protein inclusions are a common feature in Alzheimer disease (AD) and Pick disease. Even though the inclusions are morphologically different, flame-shape structure for AD vs. spherical structure for Pick disease, both have filaments mainly composed of tau protein. In AD, a well-defined pattern of conformational changes and truncation has been described. In this study, we used laser scanning confocal microscopy to characterize and compare the processing of tau protein during Pick disease with that found in AD. We found that tau protein of Pick disease preserves most of the relevant epitopes found in AD, the conformational foldings labelled by Alz-50 and Tau-66, the cleavage sites D(421) and E(391), as well as many phosphorylated sites, such as Ser(199/202), Thr(205) and Ser(396/404). We found a strong pattern of association between phosphorylation and cleavage at site D(421), as well as the phosphorylation and the conformational Alz-50 epitope. When we used late AD markers such as the conformational Tau-66 epitope and MN423 (cleavage at site E(391)) in Pick bodies (PBs), the overlap was significantly less. Furthermore, following morphological quantification, we found significantly higher numbers of phosphorylated tau in PBs. Overall, our findings suggest that phosphorylation is an early event, likely preceding the cleavage of tau at D(421). Despite this consistency with AD, we found a major distinction, namely that PBs lack beta-sheet conformation. We propose a scheme of early tau processing in these structures, similar to neurofibrillary tangles of AD.