Role of channel activation in cognitive enhancement mediated by alpha7 nicotinic acetylcholine receptors

Abstract

Background and purpose: Several agonists of the alpha7 nicotinic acetylcholine receptor (nAChR) have been developed for treatment of cognitive deficits. However, agonist efficacy in vivo is difficult to reconcile with rapid alpha7 nAChR desensitization in vitro; and furthermore, the correlation between in vitro receptor efficacy and in vivo behavioural efficacy is not well delineated. The possibility that agonists of this receptor actually function in vivo as inhibitors via desensitization has not been finally resolved.

Experimental approach: Two structurally related alpha7 nAChR agonists were characterized and used to assess the degree of efficacy required in a behavioural paradigm.

Key results: NS6784 activated human and rat alpha7 nAChR with EC(50)s of 0.72 and 0.88 microM, and apparent efficacies of 77 and 97% respectively. NS6740, in contrast, displayed little efficacy at alpha7 nAChR (<2% in oocytes, < or =8% in GH4C1 cells), although its agonist-like properties were revealed by adding a positive allosteric modulator of alpha7 nAChRs or using the slowly desensitizing alpha7V274T receptor. In mouse inhibitory avoidance (IA) memory retention, NS6784 enhanced performance as did the 60% partial agonist A-582941. In contrast, NS6740 did not enhance performance, but blocked effects of A-582941.

Conclusions and implications: Collectively, these findings suggest that a degree of alpha7 nAChR agonist efficacy is required for behavioural effects in the IA paradigm, and that such behavioural efficacy is not due to alpha7 nAChR desensitization. Also, a partial agonist of very low efficacy for this receptor could be used as an inhibitor, in the absence of alpha7 nAChR antagonists with favourable CNS penetration.

Figure 1

NS6740 and NS6784 are structurally similar α7 nicotinic acetylcholine receptor (nAChR) ligands. (A) Structures of the diazabicyclononane compounds NS6740 (A-793394) and NS6784 (A-803401). (B) Binding affinities at rat brain α7 nAChR, as measured by displacement of the α7-selective agonist [³H]A-585539. Data are shown as mean ± SEM from three experiments. Affinities of the compounds for α7 nAChR were similar, with K_i values of 1.1 nM for NS6740 and 3.3 nM for NS6784.

Figure 2

NS6784 was an agonist, while NS6740 showed little efficacy at α7 nicotinic acetylcholine receptor (nAChR) expressed in oocytes. (A) Examples of responses from human α7 nAChR expressed in oocytes. Left: NS6740 (3 µM) had little effect compared to the response to ACh (10 mM) recorded in the same oocyte. Right: NS6784 (3 µM) elicited a clear response with peak amplitude about 70% as large as the maximal response to ACh (10 mM) in the same oocyte. Lines above the traces mark the duration of compound application. Scale bars represent 1 µA and 500 ms. (B) Concentration–response curves for NS6740 and NS6784 from six oocytes expressing human α7 nAChR (left graph) and three oocytes expressing rat α7 nAChR (right graph). Responses were normalized to control responses to 10 mM ACh in each oocyte. Data are shown as mean ± SEM, and curves represent the Hill equation fit to the data. EC₅₀ values for NS6784 were 0.72 µM at human α7 nAChR, and 0.88 µM at rat α7 nAChR. NS6740 had little effect up to 100 µM.

Figure 3

NS6740 displayed weak agonist activity at human α7 nicotinic acetylcholine receptor (nAChR) expressed in GH4C1 cells. (A) An example of response of human α7 nAChR to 100 µM NS6740 (left) that was blocked by 10 nM MLA (middle). Inhibition by MLA was partially reversed following a 5 min wash (right). Lines above the traces mark duration of compound application. (B) Responses to 100 µM NS6740 were detected in 13 of 15 cells, and averaged 8.3 ± 1.9% of the response to 10 mM ACh. Lower concentrations of NS6740 had little effect, with a detectable (2%) response to 10 µM NS6740 in only one of six cells, and no detectable response to 1 µM NS6740 in six cells. (C) Interaction of NS6740 with human α7 nAChR expressed in GH4C1 cells was demonstrated by its ability to inhibit the response to 10 mM ACh. Data are shown as mean ± SEM (n= 5–7), and the curve represents the fitted Hill equation (IC₅₀= 2.7 nM).

Figure 4

Agonist activity of NS6740 revealed by α7 nicotinic acetylcholine receptor (nAChR) modulation and receptor modification. (A) At wild-type α7 nAChR, NS6740 elicited an agonist-like response in the presence of the positive allosteric modulator, PNU-120596 (10 µM). (B) Additionally, NS6740 acted as an agonist at the mutagenized α7V274T nAChR in the absence of the modulator. Responses were normalized to 10 mM ACh control responses for wild-type α7, and 10 µM ACh for α7V274T in each oocyte. Data are shown as mean ± SEM (n= 5 wild-type α7 with modulator; n= 4 with α7V274T). Curves represent the fitted Hill equation (EC₅₀ values 63 nM for α7 with modulator, 27 nM for α7V274T).

Figure 5

NS6740 inhibited responses to agonists of the α7 nicotinic acetylcholine receptor (nAChR) in the inhibitory avoidance cognitive performance model. During training, the time taken (latency) to enter the dark compartment was <20 s in all groups, as shown by the light grey bars. After training, the latency increased to 40–80 s in control saline-treated mice. Top panel: avoidance learning was enhanced by α7 nAChR agonists NS6784 (0.01–1 µmol·kg⁻¹) and A-582941 (1 µmol·kg⁻¹). Middle panel: NS6740 (0.01–10 µmol·kg⁻¹) did not enhance avoidance learning, while the positive control nicotine (0.22 µmol·kg⁻¹) did enhance learning in the same set of experiments. Bottom panel: in another group of mice, the ability of A-582941 (1 µmol·kg⁻¹) to enhance learning was blocked by prior treatment with NS6740 (0.1–10 µmol·kg⁻¹). Data are shown as mean ± SEM latency to enter the dark compartment with 10–12 mice per group. *Significantly (P < 0.05) different from saline control.