Bilateral injection of fasciculin into the amygdala of rats: effects on two avoidance tasks, acetylcholinesterase activity, and cholinergic muscarinic receptors.

These experiments examined the effects of the bilateral injection of fasciculin-2 (FAS), a natural acetylcholinesterase (AChE) inhibitory peptide, into the amygdala of rats on acquisition and retention of two avoidance behaviors. Intraamygdala injection of FAS (150 ng/amygdala) produced a pronounced and long-lasting inhibition of AChE activity: 85% and 74% on day 2 and day 5, respectively. After 48 hr, FAS-treated animals showed no changes in training or test session performance in a step-down inhibitory avoidance task (training-test interval was 24 hr). In a 2-way shuttle avoidance task, intraamygdala FAS slightly reduced retention test performance without modifying training session scores. Two and five days after FAS injections into the amygdala, the density of muscarinic receptor decreased about 50% as measured by the specific bindings of 3H-quinuclidinyl benzilate and 3H-oxotremorine. No alterations were observed in the apparent dissociation constants. On the other hand, the central-type benzodiazepine receptor population of the amygdala remained unchanged, suggesting that FAS microinjection did not produce damage to neuronal components of these nuclei. In conclusion, the results presented have indicated that a clear-cut and long-lasting inhibition of AChE activity in the amygdala is not accompanied by a facilitation of learning and memory of two different avoidance tasks. Compensation of the increased cholinergic activity by a down-regulation of muscarinic receptors could account for these findings.

Modulatory effects of phosphatidylserine on the binding of muscarinic cholinergic receptor ligands. Studies in vitro and in vivo.

The modulation of the binding of muscarinic cholinergic receptor ligands by phosphatidylserine purified from bovine cerebral cortex (BC-PS) was examined in vitro and in vivo. The enrichment of bovine cerebral cortical synaptosomal membranes with BC-PS, using a fusion technique, produced a concentration-dependent decrease in the affinity (increase in Kd) of [3H]quinuclidinyl benzylate (3H-QNB) specific binding to muscarinic acetylcholine receptors (mAChR), without changes in their maximal number (Bmax). Similar results were observed when [3H]oxotremorine (3H-OXO) was used to label a high affinity subpopulation of mAChR. On the other hand, preincubation of BC-PS liposomes with synaptosomal membranes in a nonoptimum fusion condition (at pH 7.4) did not alter the binding properties of both radioligands. Fusion experiments using a pure phosphatidylserine preparation from spinal cord revealed a similar decrement in the affinity of 3H-QNB specific binding. Five day's intraperitoneal (i.p.) administration of 15 mg/kg of BC-PS liposomes in rats increased the maximal number of cerebral cortical binding sites for 3H-OXO. Scatchard analysis revealed no changes in the apparent dissociation constant. This modification is selective in relation to the neural structure studied. Thus, BC-PS treatment did not modify 3H-OXO binding in the hippocampal formation and cerebellum. In contrast, parallel experiments using the muscarinic antagonist 3H-QNB showed no alteration in the binding properties of mAChR. Five day's i.p. administration of 15 mg/kg/d of phosphatidylcholine from bovine cerebral cortex (BC-PC) liposomes produced quite similar results to those obtained with BC-PS. These results indicate that mAChR are under the modulatory action of phosphatidylserine (PS) and phosphatidylcholine (PC), and suggest that this endogenous phospholipids may play a regulatory role on the mAChR. The possible implications of these findings on the effects of PC or PS treatment in neurological disorders involving a decrease in central cholinergic functions are discussed.

An [3H]oxotremorine binding method reveals regulatory changes by guanine nucleotides in cholinergic muscarinic receptors of cerebral cortex.

A rapid, reliable filtration method for [3H]oxotremorine binding to membranes of the cerebral cortex that allows the direct study of regulation by guanine nucleotides of muscarinic receptors was developed. [3H]Oxotremorine binds to cerebral cortex membranes with high affinity (KD, 1.9 nM) and low capacity (Bmax, 187 pmol/g protein). These sites, which represent only about 18% of those labeled with [3H]quinuclidinyl benzilate, constitute a population of GTP-sensitive binding sites. Association and dissociation binding experiments revealed a similar value of KD (2.3 nM). Displacement studies with 1-4000 nM oxotremorine showed the existence of a second binding site of low affinity (KD, 1.2 microM) and large capacity (Bmax, 1904 pmol/g protein). Gpp(NH)p, added in vitro, produced a striking inhibition of [3H]oxotremorine binding with an IC 50 of 0.3 microM. Saturation assays, in the presence of 0.5 microM Gpp(NH)p, revealed a non-competitive inhibition of the binding with little change in affinity. These results are discussed from the viewpoint of conflicting reports in the literature about guanine nucleotide regulation of muscarinic receptors in reconstituted systems and membranes from different tissues.