Various neurodegenerative diseases and psychiatric disorders are marked by alterations in brain cholinergic function and cognitive deficits. Efforts to alleviate such deficits have been limited by a lack of selective M₁ muscarinic agonists. 5-(3-Ethyl-1,2,4-oxadiazol-5-yl)-1,4,5,6-tetrahydropyrimidine hydrochloride (CDD-0102A) is a partial agonist at M₁ muscarinic receptors with limited activity at other muscarinic receptor subtypes. The present studies investigated the effects of CDD-0102A on working memory and strategy shifting in rats. CDD-0102A administered intraperitoneally 30 min before testing at 0.1, 0.3, and 1 mg/kg significantly enhanced delayed spontaneous alternation performance in a four-arm cross maze, suggesting improvement in working memory. In separate experiments, CDD-0102A had potent enhancing effects on learning and switching between a place and visual cue discrimination. Treatment with CDD-0102A did not affect acquisition of either a place or visual cue discrimination. In contrast, CDD-0102A at 0.03 and 0.1 mg/kg significantly enhanced a shift between a place and visual cue discrimination. Analysis of the errors in the shift to the place or shift to the visual cue strategy revealed that in both cases CDD-0102A significantly increased the ability to initially inhibit a previously relevant strategy and maintain a new, relevant strategy once selected. In anesthetized rats, the minimum dose required to induce salivation was approximately 0.3 mg/kg i.p. Salivation increased with dose, and the estimated ED₅₀ was 2.0 mg/kg. The data suggest that CDD-0102A has unique memory and cognitive enhancing properties that might be useful in the treatment of neurological disorders at doses that do not produce adverse effects such as salivation.

In SH-SY5Y human neuroblastoma cells, the cholinergic agonist, carbachol, stimulates phosphorylation of the small heat shock protein 27 (HSP27). Carbachol increases phosphorylation of both Ser-82 and Ser-78 while the phorbol ester, phorbol-12, 13-dibutyrate (PDB) affects only Ser-82. Muscarinic receptor activation by carbachol was confirmed by sensitivity of Ser-82 phosphorylation to hyoscyamine with no effect of nicotine or bradykinin. This response to carbachol is partially reduced by inhibition of protein kinase C (PKC) with GF 109203X and p38 mitogen-activated protein kinase (MAPK) with SB 203580. In contrast, phosphorylation produced by PDB is completely reversed by GF 109203X or CID 755673, an inhibitor of PKD. Inhibition of phosphatidylinositol 3-kinase or Akt with LY 294002 or Akti-1/2 stimulates HSP27 phosphorylation while rapamycin, which inhibits mTORC1, does not. The stimulatory effect of Akti-1/2 is reversed by SB 203580 and correlates with increased p38 MAPK phosphorylation. SH-SY5Y cells differentiated with a low concentration of PDB and basic fibroblast growth factor to a more neuronal phenotype retain carbachol-, PDB- and Akti-1/2-responsive HSP27 phosphorylation. Immunofluorescence microscopy confirms increased HSP27 phosphorylation in response to carbachol or PDB. At cell margins, PDB causes f-actin to reorganize forming lamellipodial structures from which phospho-HSP27 is segregated. The resultant phenotypic change in cell morphology is dependent upon PKC, but not PKD, activity. The major conclusion from this study is that the phosphorylated state of HSP27 in SH-SY5Y cells results from integrated signaling involving PKC, p38 MAPK and Akt.

Allosteric modulation of muscarinic acetylcholine receptors (mAChR) has been identified as a potential strategy for regulating cholinergic signaling in the treatment of various neurological disorders. Most positive allosteric modulators (PAMs) of mAChR enhance agonist affinity and potency, while very few PAMs (e.g., amiodarone) selectively enhance G protein coupling efficacy. The key structural features of amiodarone responsible for enhancement of mAChR efficacy were examined in CHO cells expressing M₁ receptors. Subsequent incorporation of these structural features into previously identified allosteric modulators of potency (i.e., n-benzyl isatins) generated ligands that demonstrated similar or better enhancement of mAChR efficacy, lower in vivo toxicity, and higher allosteric binding affinity relative to amiodarone. Notable ligands include 8a, c which respectively demonstrated the strongest binding affinity and the most robust enhancement of mAChR efficacy as calculated from an allosteric operational model. Amiodarone derivatives and hybrid ligands were additionally screened in wildtype zebrafish (Danio rerio) to provide preliminary in vivo toxicity data as well as to observe effects on locomotor and turning behaviors relative to other mAChR PAMs. Several compounds, including 8a, c, reduced locomotor activity and increased measures of turning behaviors in zebrafish, suggesting that allosteric modulation of muscarinic receptor efficacy might be useful in the treatment of repetitive behaviors associated with autism spectrum disorder (ASD) and other neuropsychiatric disorders.