Transient nicotine exposure in early adolescent male mice freezes their dopamine circuits in an immature state

Abstract

How nicotine acts on developing neurocircuitry in adolescence to promote later addiction vulnerability remains largely unknown, but may hold the key for informing more effective intervention efforts. We found transient nicotine exposure in early adolescent (PND 21-28) male mice was sufficient to produce a marked vulnerability to nicotine in adulthood (PND 60 + ), associated with disrupted functional connectivity in dopaminergic circuits. These mice showed persistent adolescent-like behavioral and physiological responses to nicotine, suggesting that nicotine exposure in adolescence prolongs an immature, imbalanced state in the function of these circuits. Chemogenetically resetting the balance between the underlying dopamine circuits unmasked the mature behavioral response to acute nicotine in adolescent-exposed mice. Together, our results suggest that the perseverance of a developmental imbalance between dopamine pathways may alter vulnerability profiles for later dopamine-dependent psychopathologies.

Fig. 1. Brief exposure to nicotine in adolescence induces a life-long imbalance between the rewarding and anxiogenic effects of later exposure.

A Top: Experimental timeline. Bottom: Timeline for the oral free-choice self-administration paradigm. B Adult mice exposed to NIC in adolescence (left) show equivalent preference for a sucrose solution as their SAC-treated counterparts. Adult mice exposed to NIC in adolescence showed a higher percentage intake of the nicotine-containing solution over all treatment doses (center). NIC-pretreated mice self-administered a higher daily dose of nicotine, with a significant difference at the 100 μg/ml dose (right, Table 1A−C). C Adult mice exposed to NIC in adulthood (left) show equivalent preference for a sucrose solution as their SAC-treated counterparts. Mice pretreated with NIC in adulthood also did not differ from controls in the percentage of nicotine solution consumed (center), nor by their daily dose of nicotine (right, Table 1D−F). D Experimental timeline. E The anxiogenic properties of an acute nicotine injection are maintained in mice pretreated with SAC in adolescence (left) but blocked in adult mice that were treated with NIC in adolescence (right, Table 1G). Graphs are separated by pre-treatment group for clarity, all statistical analyses were conducted on all four treatment conditions. F Adult mice treated with SAC in adolescence did not show CPP to a 0.2 mg/kg dose of nicotine. Adult mice treated with NIC in adolescence, however, showed CPP to this low dose of nicotine (Table 1H). G Experimental timeline. H Mice treated with NIC in adulthood show an equivalent anxiety-like response to acute nicotine (right) as their SAC-treated counterparts (left, Table 1I). Graphs are separated by pre-treatment group for clarity, but statistical analyses compared all four treatment conditions. I Mice treated with NIC or SAC as adults do not show CPP to a low dose of nicotine (Table 1J). All line graphs are presented as mean values ± SEM. Heatmaps are from representative individual animals. ^†p < 0.08, *p < 0.05, **p < 0.01, ***p < 0.01, ns = not significant. All statistical comparisons were two-sided. Holm’s sequential Bonferroni corrections were used to correct for multiple comparisons. Detailed information about statistical testing is available in Table 1. Source data are provided as a Source Data file.

Fig. 2. Nicotine in adolescence, but not in adulthood, reshapes brain-wide responsiveness to acute nicotine.

A Experimental timeline for mice treated with Nicotine (NIC; 100 μg/mL nicotine in 2% saccharin) or Saccharin (SAC; 2% saccharin only) in early adolescence or in adulthood. A’ iDISCO brain clearing and Clearmap activity mapping pipeline. B (Left) Mice treated with NIC in adolescence show greater cFos activity across the brain, and in specific brain regions, following an acute nicotine injection when compared to their SAC-treated counterparts. Overall, 129 regions showed an increase in cFos activity in NIC-pretreated mice, defined as a fold change >0 and a log p > 1.3 (equivalent to p < 0.05). Notable regions of interest associated with anxiety response and/or response to nicotine have been highlighted. (Right) The activation profile of mice treated with NIC in adulthood is not substantially different from their SAC-treated counterparts. Cell counts were compared with independent two-sample Student’s t tests assuming unequal variances, and p-values were converted to q-values to control for false discovery rate. C Correlation matrices of relationships between cFos cell numbers in response to nicotine injection. Activation across brain regions in SAC-pretreated mice (left) is highly correlated, and hierarchical clustering organizes these regions into 4 distinct modules with the strongest inter-region relationships. In NIC-pretreated mice (right), correlations between regions are less strong. Bottom, community analysis on networks formed from these correlation matrices indicate significant reorganization of the VTA-NAc-AMG connections. D Voxel-by-voxel analysis of these regions reveals increases in nicotine reactivity within the VTA and DA terminal regions. P-Value maps of significant differences between groups were generated from comparisons by independent two-sample Student’s t tests assuming unequal variances. Green or red voxels indicate a p <0.05 after FDR correction. All statistical comparisons were two-sided. Source data are provided as a Source Data file.

Fig. 3. Persistent immature neurophysiological signature of VTA dopamine neurons in mice exposed to NIC in adolescence.

A Experimental design for patch clamp experiments. B Left: No difference in peak amplitude of nicotine current between adult mice that received NIC or SAC as adolescents (NIC N = 2 mice, n = 14 neurons; SAC N = 2 mice, n = 13 neurons, Table 2A). Right: No difference in peak amplitude of nicotine current between adult mice that received NIC or SAC as adults (NIC N = 5 mice, n = 11 neurons; SAC N = 5 mice, n = 9 neurons, Table 2B). C Left: AMPA/NMDA ratio was decreased in mice that received NIC as adolescents (NIC N = 4 mice, n = 11 neurons; SAC N = 4 mice, n = 11 neurons, Table 2C). Right: NMDA current decay (weighted τ) was increased in mice that received NIC as adolescents (NIC N = 4 mice, n = 11 neurons; SAC N = 4 mice, n = 11 neurons, Table 2D). D Left: No differences in AMPA/NMDA ratio were observed when mice received NIC as adults (NIC N = 4 mice, n = 8 neurons; SAC N = 5 mice, n = 9 neurons, Table 2E). Right: NMDA current decay (weighted τ) was the same between mice that received NIC or SAC as adults (NIC N = 4 mice, n = 8 neurons; SAC N = 5 mice, n = 9 neurons, Table 2F). All bar graphs are presented as mean values ± SEM. *p < 0.05, **p < 0.01, ***p < 0.01. All statistical comparisons were two-sided. Detailed information about statistical testing is available in Table 2. Source data are provided as a Source Data file.

Fig. 4. An adolescent-like imbalance in VTA dopamine neuron response to nicotine persists in adult mice exposed to nicotine in adolescence.

A Experimental design. B Neuron responses to saline (Sal) or nicotine (Nic) injection represented as changes from baseline activity in adolescent (P28) mice (Top) and adult (>P60) mice (Bottom). Insets: Example neurons were labeled with neurobiotin (NB) and tyrosine hydroxylase (TH) to confirm their dopaminergic identity. C Nicotine evoked activation was increased in dopamine neurons of adolescent mice in comparison with adults (center, Adolescent N = 6 mice, n = 14 neurons; Adult N = 12 mice, n = 21 neurons, Table 3A), with no difference in inhibition (right, Adolescent N = 7 mice, n = 7 neurons; Adult N = 7 mice, n = 10 neurons, Table 3B). D Experimental design. E Neuron responses to Sal or Nic injection in adolescent-treated mice (left) and adult-treated mice (right). Insets: NB + /TH+ example neurons. F Nicotine evoked activation was increased in dopamine neurons of adult mice treated with NIC in adolescence in comparison with SAC-treated counterparts (center, NIC N = 29 mice, n = 70 neurons; SAC N = 24 mice, n = 70 neurons, Table 3C), with no difference in inhibition (right, NIC N = 22 mice, n = 30 neurons; SAC N = 13 mice, n = 20 neurons, Table 3D). G Nicotine evoked activation did not differ between dopamine neurons of mice treated with NIC or SAC as adults (center, NIC N = 17 mice, n = 43 neurons; SAC N = 20 mice, n = 72 neurons, Table 3E) nor in inhibition (right, NIC N = 11 mice, n = 14 neurons; SAC N = 10 mice, n = 13 neurons, Table 3F). Line graphs are presented with lines as mean values and shaded regions as ± SEM. Box plots include a box extending from the 25th to 75th percentiles, with the median indicated by a line and with whiskers extending from the minima to the maxima. *p < 0.05, **p < 0.01, ***p < 0.01. All statistical comparisons were two-sided. Detailed information about statistical testing is available in Table 3. Source data are provided as a Source Data file.

Fig. 5. Chemogenetically dampening VTA-NAc circuit activity restores anxiety-like response to nicotine in adult mice exposed to nicotine in adolescence.

A Experimental timeline for naïve mice (left). Following a 5-day CPP paradigm, adolescent mice showed a significant place preference for the chamber paired with 0.2 mg/kg nicotine (center, Table 4A), while adult mice did not (right, Table 4B). B Experimental timeline for naïve mice (left). Adolescent mice show no difference in the time spent in the open arms of the EOM between a saline or nicotine injection (center, Table 4C). Adult mice spend less time in the open arms of the EOM following an injection of nicotine than an injection of saline (right, Table 4D), indicative of an anxiogenic effect of nicotine. C Experimental timeline for DREADD intervention experiment. DAT^iCre mice were pretreated with NIC in adolescence, and then as adults they received an injection of a retroAAV hM4D(Gi) or control fluorescent-reporter virus into the NAc at the level of the medial shell. After 4 weeks, mice received an injection of CNO one hour before an injection of nicotine or saline and entering the EOM. D Retro AAV viruses were well expressed in VTA DA neurons of DAT^iCre mice following their behavioral testing (left). Mice that received a control virus replicated the effect of NIC in adolescence on WT mice, as mice never spent less time in the open arms of the EOM than their saline-treated counterparts (center, Table 4E). When VTA-NAc DA activity was reduced before nicotine injection, however, a mature behavioral response to nicotine injection, where mice spend less time in the open arms of the EOM, was restored (right, Table 4E). All line graphs are presented as mean values ± SEM. Graphs are separated by pre-treatment group for clarity, but statistical analyses compared all four treatment conditions. Heatmaps are from representative individual animals. *p < 0.05, **p < 0.01, ***p < 0.01, ns = not significant. All statistical comparisons were two-sided. Holm’s sequential Bonferroni corrections were used to correct for multiple comparisons. Detailed information about statistical testing is available in Table 4. Source data are provided as a Source Data file.