Age-dependent effects of vaping on the prefrontal cortex, ventral tegmental area, and nucleus accumbens

Abstract

Electronic nicotine delivery systems (ENDS) are unique from combustible cigarettes due to the availability of flavor options which make these devices popular among adolescents. However, there are no preclinical investigations into the impact of vaporized nicotine on late-developing brain regions such as the prefrontal cortex. Here, we investigated how neuronal function and drug self-administration differed between adult-exposed and adolescent-exposed mice. Male and female adolescent and adult C57BL/6J mice were used in a 20-session e-Vape® self-administration (EVSA) assay. Brains were then extracted and acute slices were used for either patch-clamp electrophysiology or fast-scan cyclic voltammetry. Adolescent-exposed males exhibited greater reinforcement-related behavior compared to their adult-exposed counterparts. However, adolescent-exposed and adult-exposed females exhibited similar levels of reinforcement-related behavior. Adolescent-exposed mice exhibited significant increases in intrinsic excitability of medial prefrontal cortex (mPFC) pyramidal neurons. Additionally, reinforcement-related behavior observed during EVSA assays correlated with adolescent-exposed mPFC neuronal excitability. This did not occur in adult-exposed mice. In the ventral tegmental area (VTA), we observed that upregulation of nicotinic acetylcholine receptors (nAChRs) only correlated with nicotine self-administration in adult and not adolescent-exposed mice. The relationship between self-administration and changes in neuronal excitability in adolescent mice indicates that the mPFC may be important for adolescent nicotine dependence.

Fig. 1. Adolescent and adult mouse EVSA with 60 mg/mL nicotine plus menthol.

A Schematic of EVSA paradigm. B_1–4 Active nosepokes (closed red/blue circles), inactive nosepokes (open red/blue circles), and eVape deliveries earned (closed black circles) for adult and adolescent male/female mice during the entire EVSA paradigm that passed acquisition criteria (≥2, active:inactive ratio for ≥3 sessions). C_1–4 Adult and adolescent mice assigned to PGVG only. B_1–4, C_1–4 Right axis shows number of eVape deliveries (black circles). Data are mean ± SEM. For each e-liquid condition and sex, n = 16–20 mice per group. Additional control (cue-light only) mice EVSA session data is provided in the supplemental material.

Fig. 2. Means comparison of FR3 active nosepokes and breakpoint for adult and adolescent mice that pass acquisition criteria.

A, B Mean FR3 active nosepokes and breakpoint for male adult and adolescent mice. C, D Mean FR3 active nosepokes and breakpoint for female adult and adolescent mice. Data are mean ± SEM. Data are analyzed by a Mann–Whitney U Test. *p < 0.05; ****p < 0.0001. For each condition, n = 8–14. Individual dots in each bar indicate individual mice.

Fig. 3. Adolescent-exposed mice exhibit enhanced intrinsic excitability in the mPFC.

A Representative image of a putative pyramidal neuron in the mPFC PL region in DIC imaging mode (A₂), mCherry fluorescent signal (A₃), and a representative schematic of the target brain area (A₁). B_1-3, C_1-3 Representative current-clamp waveforms from mPFC pyramidal neurons recorded from adult-exposed and adolescent-exposed mice. D_1-2 Correlation of EVSA FR3 score to rheobase of individual adolescent-exposed (D₁) or adult-exposed (D₂) mice. E_1-2 Correlation of EVSA breakpoint to rheobase of individual adolescent-exposed (E₁) or adult-exposed (E₂) mice. For D_1–2 and E_1–2, individual dots represent the mean of rheobase values for individual mice (mean of 2–3 neurons for each mouse. (D_1–2, E_1–2) For each comparison, n = 14–18 mice. Blue and red data indicate male and female mice, respectively, and open circles indicate mice assigned to control (PGVG). A_2–3, bars are 15 µm.

Fig. 4. Adolescent-exposed mice exhibit enhanced intrinsic excitability in VTA dopamine neurons.

A₁ Representative schematic of target brain region (~bregma, -3.3). A_2–5 Representative DIC and GFP image of target VTA dopamine neurons imaged on an electrophysiology setup. B_1–3, C_1–3 Representative waveforms from current-voltage protocols of neurons with different levels of intrinsic excitability. Correlation of EVSA FR3 score to rheobase (D₁) or mean breakpoint (D₂) of individual adolescent-exposed mice. E_1-2 Correlation of maximum spikes during a current step to mean FR3 score (E1) or mean breakpoint (I). Correlation of baseline firing frequency to mean FR3 score (J) or mean breakpoint (K). A_2-5 Bars, 20 µm. D_1-2, E_1-2 For each comparison, n = 14 mice. Open circles indicate mice assigned to control (PGVG), blue and red data are male and female mice, respectively.

Fig. 5. Adolescent-exposed mice exhibit enhanced baseline tonic dopamine release in the NAc core.

A Representative color plot and voltammogram of a phasic (5-pulse, 60 Hz) stimulation in the NAc core. B Representative dopamine waveform from a phasic stimulation. C Mean tonic-stimulated dopamine release in adolescent-exposed mice from PGVG or nicotine (EVSA) treatment groups. D Mean phasic-stimulated dopamine release in adult mice from PGVG or nicotine (EVSA) treatment groups. In (C, D), data were analyzed by a Mann–Whitney U Test. For each comparison, n = 5–10 mice. Data are mean ± SEM. Blue and red data are for males and females, respectively.

Fig. 6. Adult-exposed and adolescent-exposed mice exhibit differences in nicotine-induced nAChR upregulation.

A Representative schematic and 10X image of target VTA region in α4-mCherryα6-GFP mice. B Representative 20X (with 10X digital zoom) images of α6-GFP and α4-mCherry fluorescence signals in the VTA of α4-mCherryα6-GFP mice. C–N Linear regression data for VTA dopamine neuron nAChR RID of adult-exposed and adolescent-exposed mice in relation to FR3 score and breakpoint (mean of maximum active nosepokes during PR sessions). For each condition, n = 13–14 mice. Scale bar, 10 µm (B) and 100 µm (A). Correlations with “*” represent significance (p < 0.05). Colored blue and red circles are for male and female mice, respectively.

Fig. 7. Both adolescent-exposed and adult-exposed mice exhibit upregulation in VTA GABA neurons.

A Representative 20X (with 10X digital zoom) image of GABA and dopamine cells in the VTA. B–E Linear correlation of VTA GABA neuron nAChR RID with FR3 score or PR score for adult-exposed and adolescent-exposed mice. For each condition, n = 8–11 mice. Scale bar, 10 µm (A). Correlations with “*” represent significance (p < 0.05). Blue and red circles are for male and female mice, respectively.