Near‐complete adaptation of the PRiMA knockout to the lack of central acetylcholinesterase

Abstract

Acetylcholinesterase (AChE) rapidly hydrolyzes acetylcholine. At the neuromuscular junction, AChE is mainly anchored in the extracellular matrix by the collagen Q, whereas in the brain, AChE is tethered by the proline-rich membrane anchor (PRiMA).The AChE-deficient mice, in which AChE has been deleted from all tissues, have severe handicaps. Surprisingly, PRiMA KO mice in which AChE is mostly eliminated from the brain show very few deficits. We now report that most of the changes observed in the brain of AChE-deficient mice, and in particular the high levels of ambient extracellular acetylcholine and the massive decrease of muscarinic receptors, are also observed in the brain of PRiMA KO. However, the two groups of mutants differ in their responses to AChE inhibitors. Since PRiMA-KO mice and AChE-deficient mice have similar low AChE concentrations in the brain but differ in the AChE content of the peripheral nervous system, these results suggest that peripheral nervous system AChE is a major target of AChE inhibitors, and that its absence in AChE-deficient mice is the main cause of the slow development and vulnerability of these mice. At the level of the brain, the adaptation to the absence of AChE is nearly complete.

The deletion of M4 muscarinic receptors increases motor activity in females in the dark phase

“…Sixteen‐micrometer‐thick sagittal or frontal sections were cut on a cryostat at −20°C and thaw‐mounted on Superfrost ® Plus glass slides (Carl Roth GmbH & Co. KG, Karlsruhe, Germany) and stored in storage boxes at −80°C until use. For binding to MR, the sections were allowed to thaw and dry for 30 min at 22°C and the density of receptors was determined as previously described (Farar & Myslivecek, 2016; Farar et al., 2012; Valuskova, Farar, Forczek, Krizova, & Myslivecek, 2018). In brief, sections were incubated for 2 h with 2 nM [ 3 H]‐QNB at room temperature.…”

Developmental Adaptation of Central Nervous System to Extremely High Acetylcholine Levels

“…This decrease is not a subtype specific, as we observed similar reduction in pirenzepine binding (usually considered as M 1 MR, 27–51%) and AFDX-384 binding (usually considered as M 2 MR, 33–67%). Although many transmitter systems (cholinergic muscarinic and nicotinic, GABAergic, dopaminergic, glutamatergic: AMPA, kainate, NMDA) and the ACh synthesis machinery were investigated [27], we only found changes in MR and NR levels. Despite low number, MR are still responsive to cholinergic stimulation as it can be deduced from retained thermoregulatory responses to oxotremorine (MR agonist) and also preserved locomotory responses to scopolamine (MR antagonist) in PRiMA KO mice, albeit the degree of responses is changed.…”

“…Concerning the first developmental points (the determination of embryonal day and pd0), we determined these using careful mice observation. Briefly, males were allowed to cover females for five hours in the morning and after 18 days in the evening the embryos were carefully removed from uterus immediately after decapitation of dams (E18.5), the brains were removed (with cerebellum, without adjacent parts), flash frozen in liquid nitrogen and stored at −80°C until membrane fractions were prepared [27].The newborn litters were decapitated and their brains removed just after birth (pd0). To obtain remaining age points, mice were decapitated at pd9, pd30, pd120 and pd425.…”

“…Recently, we have found that in PRiMA KO mice, ACh levels are 200–300-times increased in the striatum [27], yet PRiMA KO mice are indistinguishable from (wild type) WT mice and present only marginal changes in behavior, motor skills and gait [27]. The central cholinergic system adapts to this huge increase in ACh by 20–60% decrease in MR level (accompanied by small, less than 20%, decrease in NR number).…”

Unbiased Expression Mapping Identifies a Link between the Complement and Cholinergic Systems in the Rat Central Nervous System

“…Extracellular basal levels are in the nanomolar range, but they can increase to the micromolar range after inhibiting ChEs (59,60). Also, in mutant mice partially deficient for AChE, basal extracellular levels of ACh were in the micromolar range without signs of toxicity (61). Blocking ChE activity led to effects on C3 expression at much lower ACh concentrations, suggesting that the high concentrations of ACh used initially were necessary to avoid complete depletion of ACh by ChEs present in the medium.…”