α-Conotoxin TxIB: A Uniquely Selective Ligand for α6/α3β2β3 Nicotinic Acetylcholine Receptor Attenuates Nicotine-Induced Conditioned Place Preference in Mice

Abstract

Abstract: α-Conotoxin TxIB is a specific antagonist of α6/α3β2β3(α6β2*) nicotinic acetylcholine receptor (nAChR) with an IC₅₀ of 28 nM. Previous studies have shown that α6β2* nAChRs are abundantly expressed in midbrain dopaminergic neurons and play an important role in mediating the mechanism of nicotine and other drugs reward effect. It provided important targets for the development of anti-addiction drugs. The present study evaluated the pharmacological activity of TxIB in vivo with conditioned place preference (CPP) model, which were induced by subcutaneous injection (s.c.) of nicotine (NIC, 0.5 mg/kg). α-Conotoxin TxIB inhibited the expression and reinstatement of CPP in mice dose-dependently, but had no significant effect on locomotor activity. The concentrations of dopamine (DA), γ-aminobutyric acid (GABA) and noradrenaline (NE) in different brain regions were measured by enzyme-linked immunosorbent assay (ELISA). We found that TxIB could inhibit the concentrations of DA, GABA and NE in different brain regions (such as nucleus accumbens (NAc), hippocampus (HIP) and prefrontal cortex (PFC)) in NIC-induced mice. The concentrations of DA and NE were decreased in ventral tegmental area (VTA), while GABA had little change. The current work described the inhibition activity of TxIB in NIC-induced CPP, suggesting that α6β2* nAChR-targeted compound may be a promising drug for nicotine addiction treatment.

Keywords: conditioned place preference (CPP), neurotransmitters; nAChRs; nicotine addiction.

Figure 1

The HPLC profile (A) and the MS spectrometry (B) of α-conotoxin TxIB. HPLC and MS analysis of the TxIB synthesized by two-step oxidative folding. (A) is HPLC traces of TxIB. The time (t_R) is the retention time of TxIB in HPLC profile. Analytical HPLC was carried out using an analytical Vydac C18 (5 μm, 4.6 mm × 250 mm). The separation method was as follows: a 10%–30% solvent B gradient over 30 min (solvent A is 0.1% TFA in H₂O; solvent B is 0.05% TFA in 90% ACN). (B) is MS analysis of TxIB. Its calculated mass was 1738.68 Da.

Figure 2

Schedule of conditioned place preference (CPP) experiments.

Figure 3

Effect of different doses of TxIB on locomotor activity in mice. The locomotor activity was recorded for 2 h. Data represent mean ± S.E.M. for 15 mice. There was no significance among the four groups.

Figure 4

Effect of different doses of TxIB on NIC-induced CPP expression in mice. The CPP scores (s) were considered as the time spent in the drug-paired chamber after the injection of NIC/TxIB minus the initial time spent in the drug-paired chamber. Data represent mean ± S.E.M. for 10–12 mice (n = 10–12). # indicates significant difference from the Saline + Saline group; * indicates a significant difference from the NIC + Saline group (** = p < 0.01, *** = p < 0.001, #### = p < 0.001).

Figure 5

Extinction of NIC-induced CPP. Data represent mean ± S.E.M. The 250 base-value is essentially random entry into the chamber. Natural extinction lasted for 7 days without testing. Mice were extinguished on day 5 of the training extinction. Day 1–4 differed significantly when compared with the base value. ** indicates a significant difference in the NIC group compared with the base value (** = p < 0.01, *** = p < 0.001).

Figure 6

Effect of different doses of TxIB on inhibition of CPP reinstatement in mice. Data represent mean ± S.E.M. of 10 mice per group. TxIB could inhibit reinstatement in a dose-dependently manner. * indicates a significant difference in the TxIB + NIC group compared with the Saline + NIC group (** = p < 0.01).

Figure 7

Changes of dopamine (DA), γ-aminobutyric acid (GABA) and noradrenaline (NE) in several brain regions. Data represent mean ± S.E.M. for 3–5 mice. TxIB may cause a decrease of DA, GABA and NE concentration in the nucleus accumbens (NAc), hippocampus (HIP) and prefrontal cortex (PFC) which was induced by nicotine. (a–d) represented changes of neurotransmitters in ventral tegmental area (VTA), NAc, HIP and PFC separately. In the VTA, the concentration of DA and NE were decreased, however, the concentration of GABA had few decreases. * represents a significant difference compared with the Naive group; # represents a significant difference compared to the NIC + Saline group (* = p < 0.05, ** = p < 0.01, *** = p < 0.001; # = p < 0.05, ## = p < 0.01, ### = p < 0.001).