The development of cholinergic neurons in mouse frontal cortex was studied both in vivo and in vitro by immunocytochemistry with an antibody to choline acetyltransferase (ChAT), the enzyme responsible for acetylcholine synthesis. While cortical cholinergic neurons have previously been characterized in rat cortex, up until very recently, intrinsic cortical cholinergic neurons were considered to be absent in mouse, and little is known about their development or phenotypic characteristics. The present study found no ChAT-positive neurons in mouse frontal cortex on postnatal day 0 (P0, the day of birth). On P7 there were few, faintly stained, ChAT-positive neurons. The numerical density of ChAT-positive neurons increased substantially with age, from none on P0, to 9.2 + 1.4 on P7, to 14.8 + 0.9 on P16, and 41.6 + 3.9 in adulthood. Considering that the numerical density of total neurons decreases during this postnatal period, the data represent a marked developmental increase in the percentage of cholinergic neurons. The development of cholinergic neurons showed very similar timelines in rat and mouse frontal cortex. Cultures prepared from mouse frontal cortex on embryonic day 16 were maintained for 25, 76, or 100 days in vitro (div). The percentage of ChAT-positive neurons was considerably higher than in vivo, ranging from a mean 28% to 31% across the three age (div) groups. With increasing age of the cultures, the numerical density of total neurons and ChAT-positive neurons decreased while the percentage of ChAT-positive neurons did not change significantly. These observations suggest some temporal stability in the cultures. Using dual immunofluorescence, ChAT-positive neurons were tested for colocalization with GAD or TH. The majority of ChAT-positive neurons colocalized with GAD, both in vitro and in vivo. However, ChAT did not colocalize with TH, either in vitro or in vivo. Our comparison of intact frontal cortex and cultures suggest that while the percentage of cholinergic neurons was greater in the cultures, the cholinergic neurons developed phenotypic similarities in vitro and in vivo.

Sabendo-se que o sistema colinérgico muscarínico modula funções cognitivas, propomos estudar as toxinas muscarínicas isoladas do veneno da M. lemniscatus, MT-Mlα e MT-Mlβ, sobre o processo de aprendizado e memória. Ratos Wistar machos foram injetados por via intrahipocampal com MT-Mlα, MT-Mlβ ou solução de Ringer (SRg). Após um período de sete dias de treino no Labirinto Aquático de Morris (LAM), os ratos receberam uma das toxinas ou SRg (dia da inoculação) e 20 min e 24 h após a inoculação foram testados no LAM. A MT-Mlα reduziu o tempo de permanência no quadrante do dia anterior, indicando interferência na evocação da memória. Por outro lado, a MT-Mlβ causou um efeito facilitatório quanto à recuperação da localização da plataforma. A ansiedade foi avaliada no Labirinto em Cruz Elevado, o treino foi feito após 30 min da injeção e o teste 24 h após. O tratamento com a MT-Mlβ mostrou ter um efeito ansiogênico, o que pode ter contribuído para o efeito facilitatório sobre a memória, pois sabe-se que a ansiedade até certo nível, pode favorecer o desempenho cognitivo. / Knowing that the muscarinic cholinergic system modulates cognitive functions, we propose to study the muscarinic toxins isolated from the venom of M. lemniscatus, MT-Mlα and MT-Mlβ, on the process of learning and memory. Male Wistar rats were injected by intrahippocampal pathway with MT-Mlα, MT-Mlβ or Ringer\'s solution (SRG). After a period of seven days training in Morris Water Maze (MWM), rats received a toxin or SRG (Inoculation day) and 20 min and 24 h after inoculation were tested in LAM. The MT-Mlα reduced the time spent in the quadrant of the previous day, indicating interference in the evocation of memory. On the other hand, the MT-Mlβ caused a facilitatory effect in recovering the location of the plataform. Anxiety was assessed in the Elevated Plus Maze, the training was done at 30 min after injection and 24 h after the test. Treatment with MT-Mlβ shown to have an anxiogenic effect, which may have contributed to the facilitatory effect on memory, since it is known that anxiety to a certain level can help cognitive performance.

Neuronal nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels that are necessary in memory and cognition. They are pentameric and consist of α and β subunits. They are most commonly heteromeric but, can sometimes be homomeric. nAChRs are activated by many ligands including nicotine (exogenous) and acetylcholine (endogenous).nAChRs are located on hippocampal interneurons. The interneurons, although sparse, control the synchronous firing of the pyramidal cells. However, the hippocampal interneuron structure and function is quite diverse and not fully characterized. Therefore, we sought to quantify nAChR subunit mRNA levels using real-time PCR of CA1 hippocampal interneurons.Surprisingly we found that the α3 and β2 mRNA subunits were the highest expressed and highest co-expressed subunits. Additionally, the α4 mRNA subunit was the lowest expressed of the subunits detected. The α4 subunit is one of the most pharmacologically targeted nAChR subunits and is found throughout the rest of the brain at much higher levels than the α3 mRNA subunit. Upon PCR analysis two subpopulations of the α3 and β2 subunits emerged: those that contained 3X more α3 than β2 and those that contained 3X more β2 than α3. Therefore, we hypothesized that two likely α3β2 nAChR stoichiometries are present in hippocampal interneurons. We differentiated their kinetic properties using electrophysiology.Additionally, like the α4 subunit, the α7 subunit is highly targeted in cognitive therapeutics. Since, the α7 subunit is the most characterized nAChR subunit, there are current efforts to develop allosteric modulators of the α7 subunit. The α7 subunit is found at moderate levels within hippocampal interneurons and remains a valid target. Current treatment options for Alzheimer's disease, and other dementias are limited and only mildly effective. Therefore, we sought to characterize the effect of 3-furan-2-yl-N-p-tolyl-acrylamide (PAM-2) on α7.Furthermore, there are no current methods to distinguish the α7 from the α7β2 nAChRs during whole cell electrophysiological recordings. Therefore, we also characterized the PAM-2 effect on α7β2 nAChRs. Our results highlight at least 2 ways PAM-2 can be used to differentiate α7 from the α7β2 during whole-cell recordings.

(anglický jazyk) The most common senile dementia, Alzheimer disease (AD), is characterized by a decline of memory and high cognitive functions. Typical post-mortem brain lesions are extracellular amyloid deposits, intracellular neurofibrilary tangles and ruined cholinergic and other neurotransmitters systems. Connection between damaged central cholinergic system and beta-amyloid accumulation remains obscure. We examined parietal cortex of young adult (7- month-old) female APPswe/PS1dE9 double transgenic mice which develope beta-amyloid fragments at high rate. Cholinergic synapses of these mice demonstrate functional presynaptic (stimulated acetylcholine release) as well as postsynaptic (muscarinic receptor-induced G- protein activation) deficits and reduction of cholinergic markers. The mRNA levels of choline acetyltransferase, vesicular acetylcholine transporter and M1 to M4 subtypes of muscarinic receptors were determined in transgenic and littermate controls using qPCR. Obtained experimental data does not show any changes in measured mRNA levels. These observations indicate that reduction of cholinergic synaptic markers and function is due to posttranscriptional events.

(en) Charles University in Prague Faculty of Pharmacy Hradec Králové Department of Pharmacology  Toxicology Student: Růžena Kubíčková Supervisors: Prof. Manuela Morato PharmDr. Ivan Vokřál, PhD. Title of diploma thesis: Differential reactivity of the longitudinal and circular muscle of the rat distal colon Physiological regulation of the function of the large intestine is neurohumoral. The neural part of this regulation implies the vegetative nervous system, which is crucial for the control of the gastrointestinal tract (GIT) motility. Concerning the humoral part of the regulation, recent studies showed that angiotensin II (Ang II) causes contraction of the colonic smooth muscle and, thus, can also influence the motility of the colon. However, there are no known studies that have described this process in detail. The aims of this work were (1) to compare the reactivity of the longitudinal and circular muscles of the rat distal colon to potassium chloride (KCl), acetylcholine (ACh) and Ang II, (2) to compare the observed results between male and female rats, and (3) to characterize the receptors mediating the response to Ang II. For purpose of the project, adult, 10-12 weeks old Wistar Han rats of both genders were used. Strips of the the rat distal colon were mounted in organ baths along their...

Nicotinic acetylcholine receptors are ligand-gated ion channels which are located on neuromuscular junction and in central and perifric nervous system. Activity of nicotinic receptor might be modulated by variety of pharmacological agents. In this work, we have focused on the study of the inhibition effect of tacrine and its derivatives on the nicotinic acetycholine receptors of muscle and neuronal type. These derivatives function as acetylcholinesterase inhibitors and also interact with nicotinic acetylcholine receptors. The majority of current forms of treatment of Alzheimer's disease is based on cholinesterase inhibitors. We have studied the mechanism of tacrine and its derivatives by using patch clamp method in the configuration of whole-cell recording. Powered by TCPDF (www.tcpdf.org)

Dissertation zu Expressionsmustern von muskarinergen Acetylcholin-Rezeptoren des Subtypen 1,2,3 und 4 im Gehirn von DYT1 knock-in Mäusen.

Nicotinic acetylcholine receptors (nAChR) are found widely throughout the body. Like all members of the cys-loop family of receptors, nAChRs are composed of five protein subunits, each with a large extra-cellular domain and four transmembrane domains. Together these subunits form a binding domain, transmembrane pore, and selectivity filter. Neuronal nicotinic acetylcholine receptors, formed exclusively from α2-10 and β2-4 subunits, can form in many arrangements and stoichiometries. Each arrangement can have varying binding affinities and channel kinetics, resulting in great modulatory control. α3 and β2 subunit mRNA is found in CA1 interneurons in the stratum radiatum and stratum oriens of the rat hippocampus, and in surprising expression frequency and ratios. Further study of α3 and β2 subunit mRNA injected into Xenopus laevis oocytes yields interesting results about the potential for two α3β2 subtypes. These results were in intriguing, and prompted further study to better characterize and screen the α3β2 nAChR. In order to do so, a model was needed where the α3β2 nAChR could be studied in a more physiologically relevant mammalian environment, with consistent control over α3 and β2 subunit expression ratios, and sufficient protein expression and functionality. To this end, we created a doxycycline inducible HEK-293 cell line, stably transfected with the genetic sequences for the α3 and β2 subunits and NACHO, a transmembrane protein of the neuronal endoplasmic reticulum, which has been shown to mediate the assembly of α3β2 and other nAChRs. This new model is able to induce expression various ratios between α3 and β2 subunits in a consistent, manner, proving to be valuable tool in the characterization and screening of the α3β2 nAChR.

The prokaryotic Erwinia chrysanthemi ligand-gated ion channel (ELIC) is competitively inhibited by acetylcholine (Pan et al., 2012). Acetylcholine is the native agonist of the structurally related family of eukaryotic acetylcholine receptors, which like ELIC are pentameric ligand-gated ion channels. To understand the opposite effect upon acetylcholine binding between ELIC and acetylcholine receptors, we used statistical coupling analysis to predict mutations necessary for installing acetylcholine agonism into ELIC. Statistical coupling analysis was performed on the acetylcholine binding protein from Lymnaea stagnalis. This protein is a structural surrogate for the agonist binding domain of acetylcholine receptors, for which a high-resolution structure in complex with acetylcholine is available. Our analysis identified a group of statistically coupled residues that comprises several amino acids previously implicated in acetylcholine agonism of acetylcholine receptors. Mapping these residues onto ELIC revealed 15 residue discrepancies, 4 of which were chosen for initial mutagenesis based upon their proximity to the known agonist binding site. Electrophysiological characterization of ELIC mutants indicates that the potency of the native agonist, cysteamine, is decreased, highlighting the optimized role wild-type residues serve in native agonism. None of the mutants were activated by acetylcholine, however the double mutant A75D/F133W abolished competitive antagonism by acetylcholine, and instead led to acetylcholine dependent potentiation of cysteamine-induced currents. This work demonstrates the ability of statistical coupling analysis to identify functionally important residues in pentameric ligand-gated ion channels and reveals that acetylcholine can be converted from a competitive antagonist into a potentiator, by installing two residues present in acetylcholine receptors.

Title: Study of pharmacology and function of binding sites of nicotinic acetylcholine receptors Author: Mgr. Martina Kaniaková Department: Institute of Physiology AS CR, v.v.i. Supervisor: RNDr. Jan Krůšek, CSc., Institute of Physiology AS CR, v.v.i. Abstract: Nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels. We use the whole-cell patch-clamp technique to study functional and pharmacological properties of muscle and neuronal nicotinic receptors. Rat neuronal receptors were heterologously expressed in COS cells and human embryonic muscle receptors were studied in TE671 cells. Lobeline, a plant alkaloid with a long history of therapeutic use, interacts with the classical agonist-binding site of nAChRs. The final result of this interaction depends on the receptor subtype, lobeline and other agonists concentrations and the time schedule of application. Generally, lobeline is a very weak partial agonist eliciting deep desensitization at several subtypes of nAChRs. In combination with other agonists, lobeline acts as a competitive antagonist or coagonist. Using point mutation procedure we studied the functional role of negatively charged amino acids in the F-loop of β2 and β4 subunits of neuronal receptors. Neutralising mutations in β4 subunit led to up to eighteen-fold increase in the...