Scopolamine Administration Modulates Muscarinic, Nicotinic and NMDA Receptor Systems

Studies on the effect of scopolamine on memory are abundant but so far only regulation of the muscarinic receptor (M1) has been reported. We hypothesized that levels of other cholinergic brain receptors as the nicotinic receptors and the N-methyl-D-aspartate (NMDA) receptor, known to be involved in memory formation, would be modified by scopolamine administration

Neurotransmitter receptor complexes in spatial memory and in fetal Down Syndrome brain

Es wurde gezeigt, dass seine Reihe von Rezeptoren an verschiedenen Formen von Gedächtnis Speicherung beteiligt sind. Bis jetzt gibt es aber keine Studien die die Bedeutung von Neurotransmitter Rezeptor Komplexe an räumlicher Gedächtnisspeicherung und Fötalen Down Syndrom untersucht haben, daher waren wir an solchen Studien interessiert. 1)Räumliches Gedäctnis wurde im C57BL/6J Mäusen am multiplen T-Labyrinth , einer nicht invasiven Methode getestet. 2)Der Effekt der Amnesie erzeugenden Verbindung Scopolamin wurde mit der selben Methoden verglichen. 3)Fötale Down Syndrom Gehirne wurden von einer "Gehirnbank" erhalten und für die Neurotransmiterkomplex Bestimmung verwendet. Gehirne wurden homogenisiert, Proteine extrahiert und Membranproteine wurden in Naturzustand durch Ultrazentrifugation mit einem Sucrose Gradienten erhalten. Membranproteine wurden auf nativen PAGE getrennt, auf Membrane übertragen und Immunoblotting wurde mit spezifischen Antiköroper gegen Neurotransmitter Rezeptor Untereinheiten durchgeführt. Identifikation von D1 Rezeptor Komplexe wurden durch Immunoprezipitation und anschliessender Massenspektroscopischer Identifikation der Bindungspartner im Fötalen Down Syndrome Gehirn bestimmt. AMPA Rezeptoren Komplexe mit den Untereinheiten GluA3 und GluA4 entstanden parallel zum multiplen T-Labyrinth Versuch. Scopolamine Behandlung führte zu einer Erhöhung von Rezeptor Komplexen welche Acetylcholine Muscarinisch M1, Nikotin α4 und α7, NMDA Rezeptor, GluN1, erhielten. Im männlichen und weiblichen Fötalen DS Gehirn, war D1R eniedriegt verglichen zur Kontrolle. Ein bemerkensweter Geschlechter Unterschied wurde festgestellet, da in weiblichen Down Syndrom, GluA1, GluA3, GluN1 und Nikotin Acetylcholine Rezeptor α4 und α7 erniedrigt waren. Im männlichen Down Syndrome Fötalen Gehirn waren AMPA Rezeptoren, GluA2 und GluA3 signifikant erhöht. Im Zusammenfassung zeigte sich, dass eine Serie von Neurotransmitter Rezeptoren Komplexe, parallel zur Gedächtnis Leistung und Scopolamin Amnesie moduliert wurden. Neurotransmitter Rezeptoren Veränderung findet im Fötalen Down Syndrom statt, wenn noch keine morphologischen, neurodegenerativen, oder neuropatologischen Veränderungen beobachtet werden können. Die Daten sind ein Hinweise dafür, dass Dopamine D1R Komplex ein therapeutisches Target für die Behandlung von Gedächtnisdefizierten im Down Syndrom sein könnte.A series of receptors has been shown to be involved in several forms of memory- No systematic study revealing the involvement of neurotransmitter receptor complexes, however, is available so far and we were therefore interested to study neurotransmitter receptor complexes in spatial memory as well as in fetal Down Syndrome brain. 1) Spatial memory was tested in C57BL/6j mice using the multiple-T-maze, a relatively non-invasive and well- documented paradigm 2) The effect of the amnestic compound scopolamine was tested in the same setting and 3) Fetal Down Syndrome brain was obtained from a brain bank and used for the determination of neurotransmitter receptor complexes. Brains were homogenised, proteins extracted and native membrane proteins were obtained by sucrose gradient ultracentrifugation. Membrane proteins were run on native PAGE, transferred to membranes and immunoblotting was carried out using specific antibodies against neurotransmitter receptor subunits. Identification and characterisation of a D1 containing receptor complex was carried out by immunoprecipitation and subsequent mass spectrometrical identification of binding partners in fetal Down Syndrome brains.Hippocampal AMPA receptor complexes containing subunits GluA3 and GluA4 were paralleling training in the multiple T-maze.Scopolamine administration led to an increase of receptor complexes containing acetyl choline muscarinic receptor M1, nicotinic receptors alpha 4 and 7, NMDA receptor GluN1.In fetal DS brain a receptor complex containing dopamine receptor D1R was decreased in male and female fetusses as compared to controls. A remarkable gender difference was observed as in DS females GluA1, GluA3 and GluN1 and nicotinic acetyl choline receptors alpha 4 and 7 were decreased. In male DS fetal brain AMPA receptors GluA2 and GluA3 were significantly increased. Taken together, a series of neurotransmitter receptor complexes were paralleling memory performance or modulated by the amnestic compound scopolamine. Neurotransmitter receptor changes in fetal Down Syndrome brain at a time point, when morphologically no neurodegenerative changes or Alzheimer like neuropathology is observed yet, point to an involvement of a dopamine receptor D1R containing complex, a novel finding that may indicate a possible target for experimental treatment of memory deficits in DS.submitted by Soheil Keihan FalsafiAbweichender Titel laut Übersetzung der Verfasserin/des VerfassersWien, Med. Univ., Diss., 2015OeBB(VLID)464387

Western blot results of Nic 7.

<p>Nic7 complex levels were significantly higher in scopolamine-treated groups. Significant differences were also observed when scopolamine-treated trained and untrained mice were compared.</p

Western blot results of Nic 4.

<p>Although a trend was suggested no significant differences between groups were observed.</p

Western blot results of M1.

<p>The M1 receptor complex levels were significantly higher in scopolamine-treated groups.</p

Results in the Rota rod.

<p>Significant differences in rota rod performance, in scopolamine-treated vs vehicle-treated groups (P≤0.05) are shown. Numbers are representing seconds of remaining on the revolving rod.</p

Western blot results of NR1.

<p>NR1 containing NMDA complex levels (band 1) were significantly increased in the scopolamine-treated trained group vs the vehicle-treated and trained panel (A). Band 2 (B) was not significantly different between groups.</p

Loading control.

<p>The membrane used for immunoblotting was stained by Coomassie blue R-350. Adaequate loading was shown.</p

Scheme of multiple T-maze.

<p>The multiple T-maze is a landmaze paradigm testing spatial memory. Animals with food deprivation search for food that is provided in the goal box.</p

Mass Spectrometrical Identification of Hippocampal NMDA Receptor Subunits NR1, NR2A–D and Five Novel Phosphorylation Sites on NR2A and NR2B

The NMDA receptor (NMDA-R) is a key element in neural transmission and mediating a vast variety of physiological and pathological processes in the nervous system. It is well-known that phosphorylation is required for functioning of the NMDA–R, and we therefore decided to study this post-translational modification in subunits NR1 and NR2A–D. Immunoprecipitation with an antibody against NR1 was carried out from rat hippocampi and SDS-PAGEs were run. Bands were punched, destained, and digested with trypsin and chymotrypsin and peptides were identified by nano-LC–ESI–MS/MS using an ion trap (HCT). Proteins were identified using specific software. Phosphorylations were verified by phosphatase treatment and reanalysis by mass spectrometry. The NMDA–R subunits NR1 and 2A–D were identified. On NR2A, a novel phosphorylation site was observed at S511, and on NR2B, four novel phosphorylation sites were revealed at S886, S917, S1303, and S1323 by mass spectrometry and verified by phosphatase treatment with mass spectrometrical reanalysis. A series of NMDA–R phosphorylations have been reported and these serve different functions as receptor activation, localization, and protein–protein interactions. Herein, findings of novel phosphorylation sites are extending knowledge on chemical characterization of the NMDA–R and warrant studying function of site-specific receptor phosphorylation in health and disease