Intermittent nicotine exposure upregulates nAChRs in VTA dopamine neurons and sensitises locomotor responding to the drug

Abstract

Dopaminergic projections from the ventral tegmental area (VTA) to the nucleus accumbens (NAcc) mediate the behavioral and motivational effects of many drugs of abuse, including nicotine. Repeated intermittent administration of these drugs, a pattern often associated with initial drug exposure, sensitises the reactivity of dopamine (DA) neurons in this pathway, enhances the locomotor behaviors the drugs emit, and promotes their pursuit and self-administration. Here we show that activation of nicotinic acetylcholine receptors (nAChRs) in the VTA, but not the NAcc, is essential for the induction of locomotor sensitisation by nicotine. Repeated intermittent nicotine exposure (4 × 0.4 mg/kg, base, i.p., administered over 7 days), a regimen leading to long-lasting locomotor sensitisation, also produced upregulation of nAChRs in the VTA, but not the NAcc, in the hours following the last exposure injection. Functional nAChR upregulation was observed selectively in DA but not GABA neurons in the VTA. These effects were followed by long-term potentiation of excitatory inputs to these cells and increased nicotine-evoked DA overflow in the NAcc. Withdrawal symptoms were not observed following this exposure regimen. Thus, intermittent activation and upregulation by nicotine of nAChRs in DA neurons in the VTA may contribute to the development of behavioral sensitisation and increased liability for nicotine addiction.

Figure 1

Blockade of nAChRs in the VTA (A), but not the NAcc (B), prevents locomotor sensitization by nicotine. Rats were exposed to 4 injections of nicotine (0.4mg/kg over 7 days, i.p.; ▬) or saline ( ), with or without an i.c. injection of the nAChR antagonist mecamylamine and tested 3 weeks later for their locomotor response to nicotine (0.4 mg/kg). Mecamylamine was not injected on this test. n/group=6–7. Data are shown as group mean (+SEM) 2-hr session total locomotor counts obtained after the challenge injection. Line drawings (Paxinos & Watson, 1997; numbers indicate mm from bregma) indicating location of injection cannula tips in the VTA (E) and NAcc (F) are shown for rats included in the data analyses. C. Exposure to the same regimen increased ¹²⁵I-epibatidine binding in the VTA observed at 2-hrs but not 3 days or 3 weeks following exposure. D. Significant nAChR upregulation was not observed in the NAcc at any time tested. n/group=7–10. Data (mean±SEMs) are expressed as % of saline controls (solid and dashed lines). Values for the saline control groups ranged from 90.0±15.81–130.09±10.22 fmol/mg protein in VTA and 55.68±6.96–64.62±8.91 fmol/mg protein in NAcc. No significant differences between control groups were observed in either the VTA (F_(2,24)=2.07, NS) or the NAcc (F_(2,32)=0.40, NS). *, p<0.05, **, p<0.01, compared to respective saline exposure controls.

Figure 2

Exposure to sensitizing nicotine injections (4 × 0.4mg/kg over 7 days, i.p.; ▬) leads to functional upregulation of nAChRs in VTA DA neurons and potentiation of excitatory inputs to these cells compared to saline exposed controls ( ).Traces of nAChR current responses to 300 msec focal application of 1 mM ACh onto (A) DA (large Ih) and (C) GABA-enriched (small Ih) neurons ≤120 min after the last saline or nicotine exposure injection. B. Average peak currents normalized to cell capacitance were significantly greater in DA (large Ih) neurons of nicotine exposed rats up to 2 hours following the last nicotine exposure injection. n=38,54 cells from 10,10 rats exposed to nicotine, saline, respectively. D. No significant effects of nicotine exposure were detected in GABA-enriched neurons (small Ih). n=23,25 cells from 14,15 rats. E. Traces of evoked AMPA and NMDA EPSCs. Peak AMPA current was measured at −70 mV and the NMDA current was averaged from a window 30–40 msec after the peak of the EPSC at +40 mV. F. Mean AMPAR/NMDAR ratios were significantly greater in DA (large Ih) neurons from nicotine relative to saline exposed animals. n=13,14 cells from 8,9 rats. *, p<0.05, compared to respective saline exposure controls.

Figure 3

Evoked somatic signs of withdrawal (A) and maximal nicotine-evoked NAcc DA overflow (B) following an intermittent (4 × 0.4mg/kg, i.p. over 7 days) or continuous (3.0 mg/kg/day, s.c. over 14 days) nicotine exposure regimen (▬). Compared to saline exposed controls ( ), evoked withdrawal signs were elevated 1-day following continuous but not intermittent nicotine. Nicotine-induced NAcc DA overflow was enhanced 1-day following intermittent but not continuous nicotine. n/group=5–11. Data (mean±SEMs) are expressed as % of saline-exposed controls. Values for the saline control groups were 4.5±2.1 (continuous) and 6.0±1.78 (intermittent) for number of somatic signs and 54.8±10.6 pg/25μl (continuous) and 38.5±1.7 pg/25μl (intermittent) for maximal nicotine-evoked NAcc DA overflow. No significant differences between control groups were observed for either somatic signs (t₍₁₀₎=0.54, NS) or NAcc DA (t₍₁₇₎=1.29, NS). *, p<0.05, compared to respective saline exposure controls. C. Time course for DA levels (pg/25μl) in the NAcc before and after the nicotine challenge injection (arrows at abscissae) for both exposure conditions. Numbers at abscissae indicate 20-min sampling intervals. ●, nicotine exposed; ○, saline exposed. D. Line drawings (Paxinos & Watson, 1997; numbers indicate mm from bregma) illustrating the location of the active portion of the microdialysis probes in the NAcc of rats included in the data analyses.