Enhanced nicotinic receptor function and drug abuse vulnerability

Abstract

In animals and humans, vulnerability to drug abuse varies among individuals. Animals that display high activity levels in a novel environment are more likely to self-administer psychostimulant drugs, including nicotine, cocaine, amphetamine, and morphine. Recent reports from behavioral studies indicate that nicotinic acetylcholine receptor (nAChR) activity contributes to the rewarding effects of several different addictive drugs. Thus, we hypothesized that differences in nAChR activity may contribute to the predisposition to drug self-administration. After screening of adult rats (>60 d postnatal) for the behavioral response to a novel environment, electrophysiological measures of nAChR function were conducted in brain slices that included the mesoaccumbens dopamine neurons of the ventral tegmental area (VTA). We found a positive correlation between the response to novelty and nAChR function in each assay conducted, including nAChR modulation of glutamatergic and GABAergic synaptic inputs to VTA dopamine neurons, as well as somatic nAChR responses of VTA neurons. The response to novelty and sensitivity to addictive drugs are positively correlated with the hormonal response to stress. Consistent with this observation, we found enhanced nAChR responses in vitro after a 48 h corticosterone treatment and in vivo after 48 h of repeated stress. Each of these effects was inhibited by RU486 (11beta-[p-(dimethylamino)phenyl]-17beta-hydroxy-17-(1-propynyl)estra-4,9-dien-3-one) pretreatment, suggesting a steroid hormone receptor-dependent process. These findings suggest that differences in nAChR function within the mesoaccumbens dopamine system may contribute to individual differences in drug abuse vulnerability and that these are likely attributable to differences in stress hormone levels.

Figure 1.

A, Locomotor activity in a novel environment observed in two cohorts of 12 rats. The average activity levels are plotted for the animals from the upper and lower quartiles from each activity screen (n = 6 HR and 6 LR rats). B, Schematic diagram of the horizontal slice preparation, which includes the VTA and its afferent inputs. Right is a photomicrograph of VTA neurons back labeled by injection of Fluoro-red dye into the NAcc. Scale bar, 100 μm. C, Dopamine neurons were identified based on cell diameter and the presence of an I_h current while holding at −50 mV, in response to hyperpolarizing voltage steps to −65, −80, −95, and −110 mV. Neurons with soma diameters >20 μm that expressed I_h currents >20 pA were considered to be dopaminergic. Calibration: 400 pA, 1 s.

Figure 2.

HR rats show enhanced nAChR modulation of spontaneous excitatory input to dopamine neurons of the VTA. Excitatory transmission was monitored in dopamine neurons using whole-cell voltage clamp in the presence of bicuculline (20 μm), a selective GABA_A antagonist. All synaptic transmission was recorded at −70 mV. A, Sample traces during control and nicotine treatment illustrates the increase in sEPSC frequency with bath application of nicotine (1 μm). The sample frequency histogram shows an increase in sEPSC frequency with bath-applied nicotine (1 μm). For this example, the frequency increased 233.9% of baseline activity (p < 0.05). B, The magnitude of the response to nicotine is positively correlated with locomotor activity (r = 0.641; p < 0.02; n = 15 cells from 15 animals). HR animals had a higher fraction of cells that respond to nicotine with an increase in sEPSC frequency relative to LR animals (p < 0.05). C, Basal sEPSC frequency did not correlate with locomotor activity.

Figure 3.

HR rats show enhanced nAChR modulation of spontaneous inhibitory transmission to VTA dopamine neurons. Inhibitory transmission was monitored in VTA dopamine neurons using whole-cell voltage clamp in the presence of DNQX (10 μm) to block AMPA glutamate receptors. All synaptic transmission was recorded at −70 mV. Symmetrical chloride concentrations were used to facilitate analysis, which results in inward currents with GABA_A receptor activation. A, Sample traces show sIPSCs during control and in the presence of nicotine (1 μm). The sample frequency histogram shows an increase in sIPSC frequency with bath-applied nicotine (1 μm). For this example, the sIPSC frequency increased to 215.0% of baseline (p < 0.05). After this transient increase, sIPSC frequency decreased to 64.6% of baseline (p < 0.05). B, The magnitude of the response to nicotine is positively correlated with locomotor activity (r = 0.532; p < 0.01; n = 23 cells from 23 animals). HR animals had a higher fraction of cells that respond to nicotine with an increase in sIPSC frequency relative to LR animals (p < 0.05). C, Basal sIPSC frequency did not correlate with locomotor activity.

Figure 4.

Somatic nAChR responses in the VTA correlate with locomotor activity. A, Example of a current evoked by focal pressure application of ACh (1 mm, 30 ms) to a VTA dopamine neuron in a slice from an HR rat. Calibration: 20 pA, 1 s. Nicotinic currents were isolated by TTX (1 μm), DNQX (10 μm), bicuculline (20 μm), Cd²⁺ (300 μm), and atropine (1 μm). B, Dopamine (DA) neurons in the VTA show increased nAChR function in animals with high locomotor values (r = 0.771; p < 0.05; n = 14 responses from 8 rats). C, Nondopamine neurons of the VTA showed significantly larger responses in HR animals (r = 0.674; p < 0.05; n = 18 responses from 10 rats). Data presented for both dopamine and nondopamine neurons are averages of the ACh-induced currents obtained from each animal (1–3 responses were recorded from each animal). The linear regressions were performed against the responses that elicited >3 mV depolarization according to input resistance (filled circles). Cell responses that elicited <3 mV depolarization have been plotted, as well (open squares). D, The linear regression of I_h current magnitude and locomotor index shows no significant correlation (n = 58).

Figure 5.

Chronic CORT treatment increases nicotinic receptor function in vitro. MN9D cells were differentiated for 5 d before treatment with steroid-free media alone (•), CORT (1 μm) (▴), or CORT (1 μm) and RU486 (10 μm) (■) for 1–50 h. Whole-cell voltage-clamp experiments in the absence of extracellular CORT analyzed nAChR function through focal nicotine application (100 μm) using a piezo-controlled rapid application system, with −70 mV holding potential. After chronic CORT treatment, MN9D cells showed a time-dependent increase in nicotine-induced currents (r = 0.479; p < 0.005). Nicotine-induced currents in CORT-treated cells were significantly increased after 2 d compared with age-matched controls (p < 0.007). Inhibition of the glucocorticoid receptor by RU486 blocked this increase.

Figure 6.

Stressful experiences result in increased nAChR function in LR animals through a glucocorticoid receptor-dependent mechanism. Animals subjected to a series of cold forced swim tests on 2 consecutive days were then assayed for nAChR modulation of excitatory inputs to VTA dopamine neurons. The magnitude of the increase in sEPSC frequency is significantly higher in HR than nonstressed LR animals (p < 0.05; 177.6 ± 19.5 and 118.8 ± 4.8%, respectively). Stressing LR animals led to larger nicotine responses (158.8 ± 23.4%) and eliminated the difference between HR and LR animals. In addition, RU486 (40 and 25 mg/kg on days 1 and 2, respectively) significantly reduced the effects of stress on nicotinic modulation of sEPSCs (p < 0.05; 112.5 ± 2.8%). Data are mean ± SEM; *p < 0.05.