Research Articles, Systems/Circuits

Sex Differences in the Impact of Electronic Nicotine Vapor on Corticotropin-Releasing Factor Receptor 1 Neurons in the Mouse Ventral Tegmental Area

ManHua Zhu, Neil G. Rogers, Jasmine V. Jahad and Melissa A. Herman
Journal of Neuroscience 26 April 2023, 43 (17) 3081-3093; DOI: https://doi.org/10.1523/JNEUROSCI.2087-22.2023

Abstract

Nicotine engages dopamine neurons in the ventral tegmental area (VTA) to encode reward and drive the development of nicotine addiction, however how nicotine alters a stress associated VTA population remains unclear. Here, we used male and female CRF1-GFP mice and nicotine vapor exposure to examine the effects of nicotine in VTA corticotropin-releasing factor receptor 1 (CRF1) neurons. We use immunohistochemistry and electrophysiology to examine neuronal activity, excitability, and inhibitory signaling. We found that VTA CRF1 neurons are mainly dopaminergic and project to the nucleus accumbens (NAc; VTA-NAcCRF1 neurons). VTA-NAcCRF1 neurons show greater phasic inhibition in naive females and greater focal nicotine-induced increases in firing in naive males. Following acute nicotine vapor exposure, phasic inhibition was not altered, but focal nicotine-induced tonic inhibition was enhanced in females and diminished in males. Acute nicotine vapor exposure did not affect firing in VTA-NAcCRF1 neurons, but females showed lower baseline firing and higher focal nicotine-induced firing. Activity (cFos) was increased in the CRF1 dopaminergic VTA population in both sexes, but with greater increases in females. Following chronic nicotine vapor exposure, both sexes displayed reduced basal phasic inhibition and the sex difference in tonic inhibition following acute vapor exposure was no longer observed. Additionally, activity of the CRF1 dopaminergic VTA population was no longer elevated in either sex. These findings reveal sex-dependent and exposure-dependent changes in mesolimbic VTA-NAc CRF1 neuronal activity, inhibitory signaling, and nicotine sensitivity following nicotine vapor exposure. These changes potentially contribute to nicotine-dependent behaviors and the intersection between stress, anxiety, and addiction.

SIGNIFICANCE STATEMENT Nicotine is known to engage reward systems in the brain historically centering the neurotransmitter dopamine however, how nicotine impacts other neurons in the reward pathway is less clear. The current study investigates the impact of acute and chronic electronic nicotine vapor exposure in a genetically-defined cell population containing the stress receptor corticotropin-releasing factor 1 (CRF1) that is located in the reward circuitry. This study employs functional measures of neuronal activity and identifies important sex differences in nicotine's effects across time and exposure.

Introduction

Canonically, nicotine's addictive properties involve activation of nicotinic acetylcholine receptors on dopaminergic neurons in the ventral tegmental area (VTA) driving dopamine release in the nucleus accumbens (NAc). Nicotine also produces distinct patterns of excitatory glutamatergic and inhibitory GABAergic inputs onto VTA dopamine neurons increasing overall excitability (Mansvelder and McGehee, 2000; Mansvelder et al., 2002). Although nicotine typically increases VTA dopamine neuron activity, nicotine is also inhibitory in amygdala-projecting VTA dopamine neurons and increases anxiety-like behaviors (Nguyen et al., 2021). Rodents will self-administer nicotine intravenously (Picciotto et al., 1998; O'Dell et al., 2007; Gilpin et al., 2014), intracranially into the VTA (Maskos et al., 2005; Ikemoto et al., 2006; Husson et al., 2020), and through inhalation (Cooper et al., 2021; Lallai et al., 2021). However, the impact of nicotine vapor from electronic nicotine delivery systems (ENDS) on specific VTA neuronal populations and mesolimbic reward circuitry remains unclear.

Nicotine also modulates stress and anxiety behaviors and stress-induced relapse to drug seeking (Parrott, 1993; Doherty et al., 1995; Fidler and West, 2009). The corticotropin-releasing factor (CRF) system regulates stress responding and is implicated in cocaine (Blacktop et al., 2011; Han et al., 2017; Vranjkovic et al., 2018), alcohol (Herman et al., 2016; Newman et al., 2018; Agoglia et al., 2020, 2022), and nicotine addiction (Grieder et al., 2014; Uribe et al., 2020). Nicotine activates the hypothalamic-pituitary-adrenal axis driving CRF release in the thalamus (Rohleder and Kirschbaum, 2006). Chronic nicotine exposure alters basal HPA axis activity (Rohleder and Kirschbaum, 2006) and increases CRF mRNA expression in the VTA (Grieder et al., 2014). CRF increases VTA dopamine neuron firing via CRF receptor 1 (CRF1; Wanat et al., 2008; Zalachoras et al., 2022). VTA CRF1 neurons project to the NAc core and activation of these neurons' cell bodies and terminals coordinates reward reinforcement behavior and enhance dopamine release, respectively (Heymann et al., 2020). Conversely, CRF1 deletion in midbrain dopamine neurons increases anxiety-like behavior (Refojo et al., 2011) and CRF1 antagonism reduces footshock-induced reinstatement of nicotine seeking (Bruijnzeel et al., 2009).

Nicotine's VTA effects and modulation of anxiety and stress have been well-studied, however, less is known about VTA CRF1 neurons and their response to electronic nicotine vapor, especially in females. ENDS products provide an opportunity for preclinical studies to examine sex-specific and population-specific effects of nicotine vapor exposure. We used male and female CRF1-GFP and CRF1-Cre mice to determine how acute and chronic nicotine vapor exposure dysregulates activity and inhibitory control of VTA CRF1 neurons.

Materials and Methods

Animals

Adult CRF1-GFP (Justice et al., 2008) or CRF1-Cre mice were bred in-house and group-housed in temperature-controlled and humidity-controlled 12/12 h light/dark cycle (7 A.M. lights on) facilities with ad libitum food and water access. All experimental procedures were approved by the UNC Institutional Animal Care and Use Committee.

Drugs

Freebase (-)-nicotine (N3876) and propylene glycol (PG, P4347) were purchased from Sigma. Vegetable glycerol (VG; G33-500) was purchased from Fisher. Glutamate receptor antagonists DNQX (0189) and DL-AP5 (3693) as well as GABA receptor antagonists CGP 52432 (1246) and SR 95531 (GBZ; 1262) were all purchased from Tocris Bioscience.

Immunohistochemistry

Mice were perfused and tissue sectioned and processed as previously described (Zhu et al., 2021). Sections were incubated with primary antibodies: mouse anti-TH (1:1000; T1229, Sigma), chicken anti-GFP (1:1000; ab13970, Abcam), rabbit anti-cFos (1:3000; ABE457, Millipore) and secondary antibodies: Alexa 555 goat anti-mouse (1:250; A21424, Invitrogen), Alexa 647 goat anti-mouse (1:200; 115-605-003, Jackson ImmunoResearch), Alexa 488 donkey anti-chicken (1:700; 703-545-155, Jackson ImmunoResearch) or goat anti-rabbit horse radish peroxidase (1:200; ab6721, Abcam) followed by tyramide-conjugated Cy3 (1:50) diluted in TSA amplification diluents (Akoya Biosciences, NEL741001KT). Sections were mounted (Vectashield mounting medium, H-1200, Vector Labs), cover-slipped, and imaged (Leica SP8 confocal). Quantification was performed by blinded experimenters using ImageJ (NIH).

Stereotaxic intracranial microinjection

Mice were anesthetized with isoflurane (2–4%) for stereotaxic (Kopf Instruments) intracranial infusions (100 nl/min) into target regions. CRF1-cre mice were injected with AAV5-hSyn-DIO-eGFP (500 nl/hemisphere; 50457-AAV5; titer ≥7 × 1012 vg/ml, Addgene) into the VTA (ML ±0.60, AP −3.2, DV −4.5). CRF1-GFP mice were injected with red retrograde beads (250 nl/hemisphere; Lumafluor) into the NAc (ML ±0.65, AP +1.48, DV −4.75).

Slice electrophysiology

Immediately after completion of last vapor exposure, mice were rapidly decapitated and brains extracted into sucrose solution containing (in mm): 206.0 sucrose, 2.5 KCl, 0.5 CaCl2, 7.0 MgCl2, 1.2 NaH2PO4, 26 NaHCO3, 5.0 glucose, and 5 HEPES. Coronal slices (250–300 μm) were incubated in oxygenated (95% O2/5% CO2) artificial CSF (aCSF) containing (in mm): 130 NaCl, 3.5 KCL, 2 CaCl2, 1.5 MgSO47H2O, 1.25 NaH2PO4H2O, 24 NaHCO3, and 10 glucose at 37°C (30 min) and room temperature (30 min). Recordings were performed 1–8 h following decapitation with pipettes (4–7 MΩ) filled with internal solution (in mm) 145 KCl, 5 EGTA, 5 MgCl2, 10 HEPES, 2 Na-ATP, and 0.2 Na-GTP. Spontaneous action potentials were measured in whole-cell current clamp and action potential half width and threshold to fire were quantified by Clampfit 11.1 (Molecular Devices). Rheobase was determined using current clamp with cell held at −70 mV and increments of 5- or 10-pA steps. Inhibitory transmission was measured using whole-cell voltage-clamp (Vhold = −60 mV) recording mode with glutamate receptor antagonists (20 μm DNQX and 50 μm AP5) and GABAB receptor antagonist (1 μm CGP 52432). All recordings were obtained at a sampling rate of 10,000 Hz with a Multiclamp 700B amplifier (Molecular Devices), low pass filtered at 2–5 kHz, digitized (Digidata 1440A; Molecular Devices), and stored on a computer (pClamp 10 software; Molecular Devices). Nicotine (1 μm, freebase) and gabazine, a GABAA receptor antagonist (100 μm; SR 955531) were focally applied by a y-tube positioned in close proximity to the recorded cell. Firing frequency was quantified by Clampfit. Spontaneous IPSCs (sIPSCs) from stable recording periods with ≥60 events were analyzed using MiniAnalysis (Synaptosoft). Focal nicotine (1 μm)-induced sIPSCs were analyzed for ∼2 min as previously reported (Mansvelder et al., 2002). Tonic current was determined using a Gaussian fit to an all-points histogram as previously described (Glykys and Mody, 2007).

Electronic nicotine vapor exposure

Mice were placed in airtight vacuum-controlled chambers (∼1 l/min air circulation). Vaporizers (95 W, SVS200, Scientific Vapor) heated (up to 200°C) solutions in e-vape tanks (Baby Beast Brother, Smok) with coils (0.25-Ω resistance, Smok). Mice were exposed to vaporized e-liquid solution of either 12% (v/v, or 120 mg/ml) (-)-nicotine freebase in a 50/50 (v/v) propylene glycol/vegetable glycerol (PG/VG) or PG/VG control solution (Zhu et al., 2021). E-vape controllers (SSV-1, LJARI) trigger 3 s vapor deliveries every 10 min over a 3-h session (acute) or daily 3-h sessions for 28 d (chronic).

Nicotine and cotinine serum analysis

Trunk blood was collected immediately following final vapor exposure. Samples were centrifuged and serum was collected and stored at −20°C. Samples were analyzed for nicotine and cotinine, a nicotine metabolite, using liquid chromatography-tandem mass spectrometry (LC-MS/MS) as previously described (Ghosh et al., 2019).

Statistical analysis

Data were analyzed (Prism 9.0, GraphPad) and compared using unpaired two-tailed t tests (with Welch's correction where appropriate), one-sample t tests (theoretical mean = 100), and two-way ANOVAs with post hoc Tukey's test where appropriate. Variance (F test or Bartlett's test) and normality (Kolmogorov–Smirnov test) were assessed. All data are expressed as mean ± SEM with p < 0.05 as the criterion for statistical significance.

Results

Characterization of VTA CRF1 neurons

GFP+ (CRF1) neurons were expressed predominantly in the lateral region of the VTA (Fig. 1A, top) and colocalized with tyrosine hydroxylase (TH+), a marker of dopaminergic neurons (Fig. 1A, bottom). Approximately 40% of TH+ VTA neurons co-expressed CRF1 (Fig. 1B) and ∼80% of CRF1 neurons co-expressed TH (Fig. 1C). No sex differences were observed in co-expression of either CRF1 or TH populations (Fig. 1B,C). We injected AAV-hSyn-DIO-eGFP into the VTA (Fig. 1D,E, left) of CRF1-cre mice and observed terminal expression in the NAc (Fig. 1E, right), suggesting that CRF1 neurons are part of the mesolimbic VTA-NAc circuitry.

Figure 1.
Figure 1.

Immunohistochemical characterization of VTA CRF1 neurons. A, Top, Representative image of a coronal section of the VTA; scale bar = 100 µm. Bottom, Magnified view (white box in top image) of CRF1 (GFP), tyrosine hydroxylase (TH), and merged channels; scale bar = 50 µm. B, Percentage of TH neurons in the VTA that are CRF1 positive (colored) and negative (white) in females (N = 4) and males (N = 5); unpaired t test, p = 0.67, t = 0.44, df = 7. Inset, Averaged percentage of CRF1+ neurons in female and male mice. C, Percentage of CRF1 neurons in the VTA that are TH positive (colored) and negative (white) in females (N = 4) and males (N = 5); unpaired t test, p = 0.45, t = 0.79, df = 7. Inset, Averaged percentage of TH+ neurons in female and male mice. D, Schematic of viral strategy to probe VTA CRF1 neuron projections. E, Left, VTA injection site; scale bar = 200 µm. Right, Terminal expression in the NAc; scale bar = 500 µm. Data represented as mean ± SEM. VTA- ventral tegmental area; CRF1- corticotropin releasing factor receptor 1; NAc- Nucleus accumbens.

To determine electrophysiological characteristics of VTA CRF1 neurons that project to NAc (VTA-NAcCRF1 neurons), we injected retrograde beads into the NAc of CRF1-GFP mice (Fig. 2A) and performed electrophysiological recordings in the VTA, targeting neurons co-expressing GFP (CRF1) and beads (Fig. 2A, inset). Using whole cell current clamp with no holding current, we measured spontaneous action potential characteristics in both sexes (Fig. 2B, left) and did not observe any differences in action potential shape or spiking properties. Specifically, action potential half width (Fig. 2B, right) and threshold to fire (Fig. 2C) were not significantly different between the two sexes, however rheobase was significantly higher in females as compared with males (unpaired t test *p = 0.024, t = 2.428, df = 22; Fig. 2D). Additionally, we found no significant differences in membrane properties, except increased membrane resistance in males (unpaired t test *p = 0.017, t = 2.51, df = 35.1; Fig. 2E). Voltage-clamp recordings of spontaneous IPSCs (sIPSCs; Fig. 2F) were performed and sIPSC frequency was significantly higher in females (unpaired t test *p = 0.033, t = 2.38, df = 13.4; Fig. 2G, left) but sIPSC amplitude showed no sex differences (Fig. 2G, right). Focal nicotine (1 μm) did not alter sIPSC frequency or amplitude (Fig. 2H) in either sex when normalized to baseline. Focal nicotine also induced a tonic inhibitory current in VTA-NAcCRF1 neurons of both sexes (Fig. 2I,J) that was marginal in overall magnitude but was consistently observed and was partially reversed by 100 μm gabazine (Fig. 2K). Cell-attached recordings of spontaneous firing (Fig. 2L) found no sex differences in baseline firing (Fig. 2M) but focal nicotine induced a significant (∼20%) increase in normalized firing frequency in males (*p = 0.027, t = 2.52, df = 12; Fig. 2N). These data indicate that VTA-NAcCRF1 neurons in females display greater baseline phasic inhibition, but males display greater focal nicotine-induced firing. Focal nicotine induces tonic inhibition in both sexes.

Figure 2.
Figure 2.

Electrophysiological properties and effects of focal nicotine in VTA-NAcCRF1 neurons. A, Schematic of red retrobead injection in the NAc and electrophysiological recording in the VTA in CRF1-GFP mice. Inset, Coronal section of VTA (4×) with recording electrode (right) and y-tube (left) and example of recorded neuron (60×) with differential interference contrast (DIC; top), GFP/CRF1 expression (middle), and red bead expression (bottom). B, Example traces of spontaneous action potential (AP) waveform in female (top left) and male (bottom left) and averaged action potential half width (right); unpaired t test, p > 0.05. C, Averaged threshold to fire; unpaired t test, p > 0.05. D, Averaged rheobase; unpaired t test, *p = 0.024, t = 2.428, df = 22. E, Membrane properties of VTA-NAcCRF1 neurons from female (n = 17 cells, N = 8 mice) and male (n = 28 cells, N = 11 mice) mice. Males showed higher membrane resistance than females; unpaired t test, *p = 0.017, t = 2.51, df = 35.1. F, Example traces of spontaneous IPSCs (sIPSCs) in VTA-NAcCRF1 neurons from female (left) and male (right) mice at baseline and after 1 μm focal nicotine application. G, Baseline sIPSC frequency (left) in VTA-NAcCRF1 neurons were higher in female (n = 12 cells, N = 6 mice) than male (n = 12 cells, N = 8 mice) mice; unpaired t test with Welch's correction, *p = 0.033, t = 2.38, df = 13.4. Baseline sIPSC amplitude (right) in VTA-NAcCRF1 neurons from female (n = 12 cells, N = 6 mice) and male (n = 12 cells, N = 8 mice) mice; unpaired t test, p > 0.05. H, 1 μm nicotine-induced change in sIPSC frequency (left) normalized to baseline in VTA-NAcCRF1 neurons from female (n = 10 cells, N = 5 mice) and male (n = 12 cells, N = 8 mice) mice; unpaired t test and one sample t test, p > 0.05. 1 μm nicotine-induced change in sIPSC amplitude normalized to baseline in VTA-NAcCRF1 neurons from female (n = 10 cells, N = 5 mice) and male (n = 12 cells, N = 8 mice) mice; unpaired t test and one sample t test, p > 0.05. I, Example traces of 1 μm nicotine-induced and gabazine (GBZ; 100 μm) reversed tonic current in VTA-NAcCRF1 neurons of female (top) and male (bottom) mice. J, 1 μm nicotine-induced inhibitory tonic current in VTA-NAcCRF1 neurons of female (n = 9 cells, N = 4 mice) and male (n = 9 cells, N = 6 mice) mice; unpaired t test with Welch's correction, p > 0.05. K, 100 μm gabazine (GBZ)-induced reversal of tonic current in VTA-NAcCRF1 neurons of female (n = 7 cells, N = 4 mice) and male (n = 9 cells, N = 6 mice) mice; unpaired t test, p > 0.05. L, Example traces of cell-attached firing in VTA-NAcCRF1 neurons from female (left) and male (right) mice at baseline and after 1 μm focal nicotine application. M, Baseline firing frequency in VTA-NAcCRF1 neurons of female (n = 9 cells, N = 5 mice) and male (n = 13 cells, N = 9 mice) mice; unpaired t test, p > 0.05. N, 1 μm nicotine-induced change in firing frequency normalized to baseline in VTA-NAcCRF1 neurons from female (n = 8 cells, N = 5 mice) and male (n = 13 cells, N = 9 mice; one sample t test, *p = 0.027, t = 2.52, df = 12) mice. Data are represented as mean ± SEM. VTA- ventral tegmental area; CRF1- corticotropin releasing factor receptor 1; NAc- Nucleus accumbens.

Serum nicotine metabolite levels following acute and chronic electronic nicotine vapor exposure

Acute vapor exposure consisted of a 3-h session (3-s vapor deliveries every 10 min; Fig. 3A). Mice exposed to acute 12% nicotine vapor showed significantly elevated serum nicotine (two-way ANOVA, main effect of vapor content ****p < 0.0001, F(1,40) = 32.75; PG/VG vs Nic: Female ***p = 0.0003, Male **p = 0.0055; Fig. 3B, left) and serum cotinine, the primary metabolite of nicotine (two-way ANOVA, main effect of vapor content ****p < 0.0001, F(1,40) = 53.80; PG/VG vs Nic: Female, ****p < 0.0001, Male, ****p < 0.0001; Fig. 3B, right). Chronic exposure consisted of daily 3-h sessions for 28 d (Fig. 3C). A similar effect to the acute animals, but at a larger magnitude, was observed following chronic vapor exposure where both female and male mice showed significantly elevated serum nicotine (two-way ANOVA, main effect of vapor content ****p < 0.0001, F(1,30) = 57.68; PG/VG vs Nic: Female, ****p < 0.0001, Male, ***p = 0.0002; Fig. 3D, left) and serum cotinine (two-way ANOVA, main effect of vapor content ****p < 0.0001, F(1,30) = 41.95; PG/VG vs Nic: Female, **p = 0.0087, Male, ****p < 0.0001; Fig. 3D, right). These data demonstrate that nicotine delivery through electronic vapor is an effective route of administration that produces significant serum nicotine and cotinine levels following both acute and chronic exposure, however serum levels were more elevated following chronic exposure suggesting potential impairment of nicotine metabolism.

Figure 3.
Figure 3.

Nicotine and cotinine serum levels following acute and chronic electronic nicotine vapor exposure. A, Acute vapor exposure paradigm depicting a 3-h session of 3-s vape every 10 min. B, Serum nicotine (left; two-way ANOVA, main effect of vapor content ****p < 0.0001, F(1,40) = 32.75, post hoc Tukey's test: Female PG/VG vs Nic ***p = 0.0003, Male PG/VG vs Nic **p = 0.0055) and serum cotinine (right; two-way ANOVA, main effect of vapor content ****p < 0.0001, F(1,40) = 53.80, post hoc Tukey's test: Female PG/VG vs Nic ****p < 0.0001, Male PG/VG vs Nic ****p < 0.0001) levels following acute PG/VG or 12% nicotine vape exposure in female and male mice. C, Chronic vapor exposure paradigm depicting daily 3-h sessions of 3-s vape every 10 min over the course of 28 d. D, Serum nicotine (left; two-way ANOVA, main effect of vapor content ****p < 0.0001, F(1,40) = 43.42, post hoc Tukey's test: Female PG/VG vs Nic ****p < 0.0001, Male PG/VG vs M. Nic **p = 0.0013) and serum cotinine (right; two-way ANOVA, main effect of vapor content ****p < 0.0001, F(1,40) = 37.20, post hoc Tukey's test: Female PG/VG vs F Nic **p = 0.0071, Male PG/VG vs Nic ****p < 0.0001) levels following chronic PG/VG or 12% nicotine vape exposure in female and male mice. **p < 0.005, ***p < 0.0005, ****p < 0.0001. Data are represented as mean ± SEM.

The effects of acute vapor exposure on inhibitory signaling in VTA-NAcCRF1 neurons

We exposed CRF1-GFP mice to acute PG/VG or 12% nicotine vapor (Fig. 3A) and performed slice electrophysiology targeting VTA-NAcCRF1 neurons using bead injections as described above (Fig. 2A). We found no significant difference in action potential half width (two-way ANOVA, p > 0.05; Fig. 4A, left) or threshold to fire between groups (two-way ANOVA, p > 0.05, Female PG/VG −40.2 ± 1.37, Female Nic −38.0 ± 1.28, Male PG/VG −39.6 ± 1.27, Male Nic −39.9 ± 0.73 mV). Rheobase showed a significant sex × vapor content interaction (two-way ANOVA, *p = 0.049, F(1,51) = 4.073; Female PG/VG 35.7 ± 5.57, Female Nic 50.4 ± 7.14, Male PG/VG 59.7 ± 9.54, Male Nic 42.7 ± 7.42 pA) but no significant main effects were observed and post hoc Tukey's test did not yield any significant comparisons. Additionally, we found no significant differences in membrane properties (Fig. 4A, right), except membrane capacitance (main effect of vapor content *p = 0.035, F(1,77) = 4.59, but p > 0.05 by Tukey's multiple comparisons test). In females (Fig. 4B) and males (Fig. 4C), there were no significant differences in baseline sIPSC frequency or amplitude (Fig. 4D). In both sexes, focal nicotine (1 μm) produced no change in sIPSC frequency when normalized to baseline (Fig. 4E, left). However, normalized sIPSC amplitude in 12% nicotine-exposed males was significantly increased (one sample t test *p = 0.011, t = 3.41, df = 7; Fig. 4E, right). Focal nicotine (1 μm) induced a tonic current that was comparable in PG/VG groups but enhanced in 12% nicotine-exposed females and reduced in 12% nicotine-exposed males (sex × vapor content interaction **p = 0.005, F(1,29) = 9.49; main effect of sex *p = 0.039, F(1,29) = 4.68; Female: PG/VG vs Nic *p = 0.024, Female Nic vs Male Nic **p = 0.0091; Fig. 4F,G). Gabazine (GBZ; 100 μm) partially reversed the focal nicotine-induced tonic current (Fig. 4H). These data suggest that phasic inhibition in VTA-NAcCRF1 neurons is largely unaffected but focal nicotine-induced tonic inhibition was bidirectionally dysregulated in a sex-specific manner following acute nicotine vapor exposure.

Figure 4.
Figure 4.

Inhibitory signaling in VTA-NAcCRF1 neurons following acute vapor exposure. A, Averaged action potential half width (left) and membrane properties (right) of VTA-NAcCRF1 neurons from male and female mice exposed to acute PG/VG (Female n = 23 cells, N = 6 mice; Male n = 22 cells, N = 5 mice) or 12% nicotine vapor (Female n = 15 cells, N = 4 mice; Male n = 21 cells, N = 5 mice) with a main effect of vapor content on membrane capacitance (two-way ANOVA *p = 0.035, F(1,77) = 4.59). B, Example of spontaneous IPSCs (sIPSCs) in VTA-NAcCRF1 neurons from female mice exposed to PG/VG (left) or 12% nicotine (right) at baseline and after 1 μm nicotine application. C, Example of sIPSCs in VTA-NAcCRF1 neurons from male mice exposed to PG/VG (left) or 12% nicotine (right) at baseline and after 1 μm nicotine application. D, Baseline sIPSC frequency (left) and amplitude (right) in VTA-NAcCRF1 neurons from female and male mice exposed to PG/VG (Female n = 11 cells, N = 5 mice; Male n = 11 cells, N = 4 mice) or 12% nicotine (Female n = 10 cells, N = 4 mice; Male n = 8 cells, N = 5 mice); two-way ANOVA p > 0.05. E, 1 μm nicotine-induced change in sIPSC frequency (left) and amplitude (right) normalized to baseline in VTA-NAcCRF1 neurons from female and male mice exposed to PG/VG (Female n = 9 cells, N = 5 mice; Male n = 11 cells, N = 4 mice) or 12% nicotine (Female n = 9 cells, N = 4 mice; Male n = 8 cells, N = 5 mice). Normalized sIPSC amplitude show vapor content × sex interaction (two-way ANOVA *p = 0.010, F(1,33) = 7.45) and specifically, the male 12% Nic group shown significant increase from baseline (one sample t test *p = 0.011, t = 3.41, df = 7). F, Example traces of 1 μm nicotine-induced and gabazine (GBZ) reversed tonic current in VTA-NAcCRF1 neurons from female (top) and male (bottom) mice exposed to PG/VG (left) or 12% nicotine (right). G, 1 μm nicotine-induced tonic current in VTA-NAcCRF1 neurons from female and male mice exposed to PG/VG (Female n = 9 cells, N = 5 mice; Male n = 10 cells, N = 4 mice) or 12% nicotine (Female n = 8 cells, N = 4 mice; Male n = 6 cells, N = 3 mice). Tonic current showed a vapor content × sex interaction (**p = 0.005, F(1,29) = 9.49), main effect of sex (*p = 0.039, F(1,29) = 4.68) and post hoc Tukey's significance test in Female: PG/VG versus Nic (*p = 0.024) and Female Nic versus Male Nic (**p = 0.0091) by two-way ANOVA. H, Gabazine (GBZ; 100 μm) reversal of tonic current in VTA-NAcCRF1 neurons of female and male mice exposed to PG/VG (Female n = 8 cells, N = 4 mice; Male n = 7 cells, N = 4 mice) or 12% nicotine (Female n = 8 cells, N = 4 mice; Male n = 6 cells, N = 3 mice); two-way ANOVA p > 0.05. Data are represented as mean ± SEM.

The effects of acute vapor exposure on spontaneous firing of VTA-NAcCRF1 neurons

We performed cell-attached recordings in female (Fig. 5A) and male (Fig. 5B) mice exposed to acute PG/VG or 12% nicotine vapor. We observed higher baseline firing frequency in males as compared with females (main effect of sex *p = 0.028, F(1,49) = 5.11; Fig. 5C), however with a marginal overall effect size. Focal nicotine (1 μm)-induced changes in firing normalized to baseline was significantly higher in females exposed to either PG/VG or 12% nicotine as compared with males (main effect of sex *p = 0.025, F(1,37) = 5.44; Fig. 5D). These data suggest that acute exposure does not affect spontaneous firing in VTA-NAcCRF1 neurons. However, females exposed to either PG/VG or 12% nicotine vapor show lower baseline firing and focal nicotine-induced increases in firing.

Figure 5.
Figure 5.

Spontaneous firing in VTA-NAcCRF1 neurons following acute vapor exposure. A, Example of spontaneous firing in VTA-NAcCRF1 neurons from female mice exposed to acute PG/VG (left) or 12% nicotine (right) at baseline and after 1 μm focal nicotine application. B, Example of spontaneous firing in VTA-NAcCRF1 neurons from male mice exposed to PG/VG (left) or 12% nicotine (right) at baseline and after 1 μm nicotine application. C, Baseline firing in VTA-NAcCRF1 neurons from females and males exposed to acute PG/VG (Female n = 16 cells, N = 5 mice; Male n = 10 cells, N = 4 mice) or 12% nicotine (Female n = 13 cells, N = 5 mice; Male n = 14 cells, N = 4 mice); two-way ANOVA, main effect of sex *p = 0.028, F(1,49) = 5.11. D, 1 μm nicotine-induced change in firing in VTA-NAcCRF1 neurons from female and male mice exposed to acute PG/VG (Female n = 10 cells, N = 5 mice; Male n = 9 cells, N = 4 mice) or 12% nicotine (Female n = 9 cells, N = 5 mice; Male n = 13 cells, N = 4 mice); two-way ANOVA, main effect of sex *p = 0.025, F(1,37) = 5.44. Data are represented as mean ± SEM.

The effects of acute vapor exposure on VTA subpopulation activity

To examine overall activity in the VTA we performed immunohistochemistry to label cFos, tyrosine hydroxylase (TH), and CRF1 (Fig. 6A) following acute vapor exposure as described above (Fig. 3A). Animals of both sexes exposed to acute 12% nicotine vapor showed an overall increase in number of cFos+ neuron expression in the VTA (main effect of vapor content *p = 0.022, F(1,13) = 6.80; Fig. 6B). Specifically, cFos expression was increased in the TH+ (dopaminergic) VTA population in both sexes exposed to 12% nicotine compared with PG/VG (main effect of vapor content ***p = 0.0002, F(1,13) = 27.47; post hoc Tukey's test: Female PG/VG vs Nic *p = 0.014, Male PG/VG vs Nic *p = 0.011; Fig. 6C). cFos expression in the CRF1+ VTA population was significantly higher in acute 12% nicotine-exposed mice as compared with PG/VG-exposed mice (main effect of vapor content *p = 0.013, F(1,13) = 8.35); however, post hoc tests did not pull out any significant comparisons (Fig. 6D). cFos expression in TH+ and CRF1+ co-expressing neurons was also increased in 12% nicotine vapor-exposed animals (main effect of vapor content *p = 0.013, F(1,13) = 8.28), but post hoc comparisons were only significant for females (PG/VG vs Nic *p = 0.048; Fig. 6E). Overall, these data indicate that acute nicotine vapor exposure increases activity in VTA dopamine and VTA CRF1 neuronal populations of both sexes but with more profound effects in females.

Figure 6.
Figure 6.

Changes in population activity in the VTA following acute vapor exposure. A, Representative images of cFos, tyrosine hydroxylase (TH), CRF1 expression, and merged images of the VTA of a female nicotine vapor exposed mouse; scale bar = 50 µm. B, Number of cFos+ neurons in the VTA in females and males exposed to acute PG/VG (Female N = 4, Male N = 4) or acute 12% nicotine (Female N = 4, Male N = 5); two-way ANOVA, main effect of vapor content *p = 0.022, F(1,13) = 6.80. C, Number of VTA neurons that express cFos and TH in females and males exposed to acute PG/VG (Female N = 4, Male N = 4) or acute 12% nicotine (Female N = 4, Male N = 5); two-way ANOVA, main effect of vapor content ***p = 0.0002, F(1,13) = 27.47, post hoc Tukey's test: Female PG/VG versus Nic *p = 0.014, Male PG/VG versus Nic *p = 0.011. D, Number of VTA neurons that express cFos and CRF1 in females and males exposed to acute PG/VG (Female N = 4, Male N = 4) or acute 12% nicotine (Female N = 4, Male N = 5); two-way ANOVA, main effect of vapor content *p = 0.013, F(1,13) = 8.35, two-way ANOVA). E, Number of VTA neurons that express cFos, TH, CRF1 in females and males exposed to acute PG/VG (Female N = 4, Male N = 4) or acute 12% nicotine (Female N = 4, Male N = 5); two-way ANOVA, main effect of vapor content *p = 0.013, F(1,13) = 8.28, post hoc Tukey's test: Female PG/VG versus Nic *p = 0.048. Data are represented as mean ± SEM.

The effect of chronic vapor exposure on inhibitory signaling in VTA-NAcCRF1 neurons

VTA-NAcCRF1 neurons from CRF1-GFP mice exposed to chronic vapor (as described in Fig. 3C) display no significant differences in action potential half width between groups (two-way ANOVA, p > 0.05; Fig. 7A, left) but there was a significant main effect of sex (two-way ANOVA, *p > 0.042, post hoc Tukey's test p > 0.05) in threshold to fire (Female PG/VG −41.6 ± 1.43, Female Nic −40.2.0 ± 1.40, Male PG/VG −39.2 ± 0.90, Male Nic −37.8 ± 0.88). Rheobase (two-way ANOVA, p > 0.05, Female PG/VG 57.4 ± 10.2, Female Nic 50.5 ± 6.04, Male PG/VG 63.0 ± 5.59, Male Nic 54.6 ± 7.59) and other membrane properties (Fig. 7A, right) were also not significantly different across all groups. sIPSCs were recorded from females (Fig. 7B) and males (Fig. 7C) exposed to chronic PG/VG or chronic 12% nicotine vapor. We found that baseline sIPSC frequency was lower in 12% nicotine-exposed mice (main effect of vapor content *p = 0.039, F(1,52) = 4.51), likely driven by males; however, post hoc Tukey's test was not significant between groups (Fig. 7D, left). Baseline sIPSC amplitude was not altered in either sex exposed to PG/VG or 12% nicotine (Fig. 7D, right). When 1 μm nicotine-induced changes in sIPSC frequency and amplitude were normalized to baseline, there were no significant differences between sexes from either PG/VG or 12% nicotine groups (Fig. 7E). Focal nicotine induced a tonic inhibitory current in VTA-NAcCRF1 neurons of both sexes exposed to either PG/VG or 12% nicotine (Fig. 7G) that was partially reversed with 100 μm gabazine (Fig. 7H). These data suggest that chronic nicotine vapor exposure reduced presynaptic phasic inhibition in VTA-NAcCRF1 neurons from males and the sex difference in tonic inhibition observed following acute nicotine vapor exposure was no longer present.

Figure 7.
Figure 7.

Inhibitory signaling in VTA-NAcCRF1 neurons following chronic vapor exposure. A, Averaged action potential half width (left) and membrane properties of VTA-NAcCRF1 neurons from male and female mice exposed to chronic PG/VG (Female n = 24 cells, N = 4 mice; Male n = 33 cells, N = 6 mice) or 12% nicotine vapor (Female n = 25 cells, N = 5 mice; Male n = 32 cells, N = 6 mice). B, Examples of spontaneous IPSCs (sIPSCs) in VTA-NAcCRF1 neurons from female mice exposed to PG/VG (left) or 12% nicotine (right) at baseline and after 1 μm nicotine application. C, Examples of spontaneous IPSCs (sIPSCs) in VTA-NAcCRF1 neurons from male mice exposed to PG/VG (left) or 12% nicotine (right) at baseline and after 1 μm nicotine application. D, Baseline sIPSC frequency (left; two-way ANOVA, main effect of vapor content *p = 0.039, F(1,52) = 4.51) and amplitude (right; two-way ANOVA, p > 0.05) from female and male mice exposed to either PG/VG (Female n = 13 cells, N = 4 mice; Male n = 16 cells, N = 6 mice) or 12% nicotine (Female n = 12 cells, N = 5 mice; Male n = 16 cells, N = 6 mice). E, 1 μm nicotine-induced change in sIPSC frequency (left) and amplitude (right) normalized to baseline in VTA-NAcCRF1 neurons from female and male mice exposed to PG/VG (Female n = 11 cells, N = 4 mice; Male n = 14 cells, N = 6 mice) or 12% nicotine (Female n = 9 cells, N = 5 mice; Male n = 14 cells, N = 6 mice); two-way ANOVA and one sample t test, p > 0.05. F, Example traces of 1 μm nicotine-induced and gabazine (GBZ) reversed tonic current in VTA-NAcCRF1 neurons from female (top) and male (bottom) mice exposed to PG/VG (left) or 12% nicotine (right). G, 1 μm nicotine-induced tonic current in VTA-NAcCRF1 neurons from female and male mice exposed to PG/VG (Female n = 10 cells, N = 3 mice; Male n = 13 cells, N = 6 mice) or 12% nicotine (Female n = 8 cells, N = 5 mice; Male n = 14 cells, N = 6 mice); two-way ANOVA, p > 0.05. H, Gabazine (GBZ; 100 μm) reversal of tonic current in VTA-NAcCRF1 neurons from female and male mice exposed to PG/VG (Female n = 9 cells, N = 3 mice; Male n = 12 cells, N = 6 mice) or 12% nicotine (Female n = 8 cells, N = 5 mice; Male n = 13 cells, N = 5 mice); two-way ANOVA, p > 0.05. Data are represented as mean ± SEM.

The effect of chronic vapor exposure on spontaneous firing of VTA-NAcCRF1 neurons

We performed cell-attached recordings in female (Fig. 8A) and male (Fig. 8B) mice exposed to chronic PG/VG or 12% nicotine vapor and observed no differences in baseline firing in either vapor condition (Fig. 8C). When normalized to baseline, focal nicotine (1 μm) did not significantly change spontaneous firing in either sex or vapor group (interaction *p = 0.045, F(1,38) = 4.29; Fig. 8D). These data suggest that sex differences in baseline firing and effects of focal nicotine on firing in VTA-NAcCRF1 neurons following acute exposure is reversed following chronic exposure to PG/VG or 12% nicotine vapor.

Figure 8.
Figure 8.

Spontaneous firing in VTA-NAcCRF1 neurons following chronic vapor exposure. A, Example of spontaneous firing in VTA-NAcCRF1 neurons from female mice exposed to chronic PG/VG (left) or 12% nicotine (right) at baseline and after 1 μm focal nicotine application. B, Example of spontaneous firing in VTA-NAcCRF1 neurons from male mice exposed to chronic PG/VG (left) or 12% nicotine (right) at baseline and after 1 μm focal nicotine application. C, Baseline firing in VTA-NAcCRF1 neurons from females and males exposed to chronic PG/VG (Female n = 10 cells, N = 4 mice; Male n = 13 cells, N = 5 mice) or 12% nicotine (Female n = 13 cells, N = 5 mice; Male n = 17 cells, N = 5 mice); two-way ANOVA, p > 0.05. D, 1 μm nicotine-induced change in firing in VTA-NAcCRF1 neurons from female and male mice exposed to chronic PG/VG (Female n = 7 cells, N = 4 mice; Male n = 12 cells, N = 5 mice) or 12% nicotine (Female n = 11 cells, N = 5 mice; Male n = 12 cells, N = 4 mice); two-way ANOVA, interaction *p = 0.045, F(1,38) = 4.29. Data are represented as mean ± SEM.

The effect of chronic vapor exposure on VTA subpopulation activity

To examine overall activity in the VTA following long-term exposure to vapor, we exposed animals to either PG/VG or 12% nicotine chronically (as described in Fig. 3C) and performed immunohistochemistry to label cFos, tyrosine hydroxylase (TH), and CRF1 (Fig. 9A). We found that animals from both sexes exposed to chronic 12% nicotine vapor show overall increased number of cFos+ neurons (main effect of vapor content *p = 0.045, F(1,15) = 4.80, Fig. 9B). cFos expression in the TH+ VTA population was increased in 12% nicotine-exposed mice compared with PG/VG-exposed mice (main effect of vapor content *p = 0.010, F(1,15) = 8.62), and post hoc tests showed a significant difference between vapor groups in females but not males (Female: PG/VG vs nicotine *p = 0.044; Fig. 9C). There was no difference in cFos expression in the CRF1+ or the TH+/CRF1+ VTA population in either sex from both vapor groups (Fig. 9D,E). These data indicate that chronic nicotine vapor exposure maintains elevated activity in VTA dopaminergic neurons, especially in females. However, the increased activity in CRF1+ neurons following acute exposure is reduced to levels similar to PG/VG after chronic exposure.

Figure 9.
Figure 9.

Changes in population activity in the VTA following chronic vapor exposure. A, Representative image of cFos, tyrosine hydroxylase (TH), CRF1 expression, and merged images of the VTA of a male nicotine vapor exposed mouse; scale bar = 50 µm. B, Number of cFos+ neurons in the VTA in females and males exposed to chronic PG/VG (Female N = 4, Male N = 4) or chronic 12% nicotine (Female N = 4, Male N = 5); two-way ANOVA, main effect of vapor content *p = 0.045, F(1,15) = 4.80. C, Number of VTA neurons that express cFos and TH in females and males exposed to chronic PG/VG (Female N = 6, Male N = 4) or chronic 12% nicotine (Female N = 5, Male N = 4); two-way ANOVA, main effect of vapor content *p = 0.010, F(1,15) = 8.62, post hoc Tukey's test: Female PG/VG versus nicotine *p = 0.044. D, Number of VTA neurons that express cFos and CRF1 in females and males exposed to chronic PG/VG (Female N = 6, Male N = 4) or chronic 12% nicotine (Female N = 5, Male N = 4); two-way ANOVA, p > 0.05. E, Number of VTA neurons that express cFos, TH, CRF1 in females and males exposed to chronic PG/VG (Female N = 6, Male N = 4) or chronic 12% nicotine (Female N = 5, Male N = 4); two-way ANOVA, p > 0.05. Data are represented as mean ± SEM.

Discussion

VTA CRF1 neurons are predominately dopaminergic and project to the NAc, suggesting involvement in the mesolimbic reward pathway. There are sex differences in phasic inhibition and nicotine-induced firing in VTA-NAcCRF1 neurons, but both sexes displayed focal nicotine-induced tonic inhibition. Following acute nicotine vapor exposure, phasic inhibition and firing in VTA-NAcCRF1 neurons were largely unaffected, but focal nicotine-induced tonic inhibition was enhanced in females and reduced in males. Activity of the CRF1 dopaminergic VTA population increased in both sexes following acute nicotine vapor exposure, but especially in females. Chronic nicotine vapor exposure reduced phasic inhibition in VTA-NAcCRF1 neurons from both sexes and the sex-specific bidirectional changes in tonic inhibition were no longer observed. Additionally, activity of the CRF1 dopaminergic VTA population was no longer elevated following chronic nicotine vapor exposure. Collectively, these findings demonstrate sex-specific differences in inhibitory control and response to focal nicotine in VTA-NAcCRF1 neurons from naive mice, and sex-specific changes in activity and inhibitory control following acute nicotine vapor exposure that were lost following chronic exposure. These findings identify important sex-dependent and exposure-dependent changes in mesolimbic CRF1 population activity and how electronic nicotine vapor selectively impacts reward and stress circuitry in males and females.

Electronic nicotine delivery systems (ENDS) use has increased dramatically, however, the effects of nicotine vapor on brain reward and stress circuitry remain unclear. Previous studies have used alternative forms of nicotine delivery like subcutaneous minipumps (Salas et al., 2009; Grieder et al., 2014), drinking water (DeBaker et al., 2020; Wong et al., 2020), intraperitoneal (Pauly et al., 1992; Caruso et al., 2018; Akers et al., 2020), or intravenous (Picciotto et al., 1998; O'Dell et al., 2007) injections. This study used a nicotine vapor inhalation model that allows nicotine to reach the brain on a timescale similar to nicotine from cigarettes (Solingapuram Sai et al., 2020), which is a primary determinant of nicotine reinforcement (Henningfield and Keenan, 1993). Additionally, vapor liquids can contain higher nicotine concentrations than traditional cigarettes, which may cause differential engagement of the reward circuitry. Our previous study found that a single 3 s 12% nicotine vapor produced mouse serum nicotine levels (Zhu et al., 2021) that are comparable to human serum levels in cigarette smokers (Benowitz et al., 1988; Chellian et al., 2021) and electronic cigarette users (St Helen et al., 2016). Additionally, serum cotinine levels in mice following acute nicotine vapor exposure (Fig. 3B, right) were comparable to those found in humans who smoke cigarettes daily or use e-cigarettes daily (Rapp et al., 2020). However, with repeated nicotine vapor administrations (acute and chronic exposure paradigms), we did observe serum nicotine levels higher than what have been reported in humans. This discrepancy can be because of a variety of potential factors including the species' different nicotine metabolism rates (mice have faster nicotine metabolism than humans), route of inhalation and absorption (mice inhale the vapor mainly through the nose whereas humans inhale vapor through the mouth), time of sample collection (mouse samples in our study collected immediately following last vape session vs timing of human sample collection can vary depending on the study), and naturalistic mouse behavior (potential ingestion of nicotine deposits on fur when grooming). Future studies will examine nicotine's effects at lower concentrations as well as incorporating self-administration to better model animal's motivation to seek nicotine.

Clinical studies suggest that women, more than men, report stress as a major factor promoting nicotine use (Fidler and West, 2009). Sex differences in nicotine metabolism (Johnstone et al., 2006; Kandel et al., 2007) could also contribute to neurobiological effects, highlighting the gap in knowledge on sex-specific effects and the need for female subjects in preclinical research. We examined VTA-NAcCRF1 neurons in both sexes and found that females displayed higher basal phasic inhibition and enhanced tonic inhibition after exposure to acute nicotine vapor compared with males. These data indicate that female VTA CRF1 neurons are under greater inhibitory control which may explain female-specific decreases in dopamine release in the nucleus accumbens with administration of the nicotinic receptor antagonist, mecamylamine (Carcoba et al., 2018). Overexpression of CRF in the nucleus accumbens has also been shown to enhance the reinforcing effects of nicotine in females (Uribe et al., 2020). Future experiments examining the effects of CRF in both female and male VTA-NAcCRF1 neurons will shed light on how stress affects reward processing in the VTA.

We found that ∼80% of the CRF1 neurons in the VTA are dopaminergic which means that the remaining ∼20% are nondopaminergic and can potentially overlap with other cell types of the VTA. Given that the VTA CRF1 population is mainly expressed in the lateral VTA it is likely that the remaining 20% are primarily GABAergic however, CRF1 neurons located in the most medial region of the lateral VTA may also be glutamatergic or represent a mixed transmitter population. In naive males, we observed an increase in firing induced by application of 1uM of nicotine, consistent with previous studies from VTA dopamine neurons in slice recordings (Pidoplichko et al., 1997; Picciotto et al., 1998; Mansvelder and McGehee, 2000; Yin and French, 2000). However, following administration of nicotine intravenously, in vivo single-cell extracellular recording from VTA dopamine neurons found that nicotine produces increased firing in some and decreased firing in others (Eddine et al., 2015; Nguyen et al., 2021). This inhibition required D2 receptor activation (Eddine et al., 2015) and was found in the amygdala-projecting population to mediating anxiety-like behavior (Nguyen et al., 2021). These discrepancies in nicotine's effect in the VTA may be because of differences in route of nicotine administration (in vivo vapor vs in vivo intravenous vs in vitro bath application) and cell type population in the VTA (dopamine/GABA/Glut or projection specific). Following acute nicotine vapor exposure, VTA CRF1 neuronal activity as measured by cFos was increased in both sexes but neither sex showed increases in baseline spontaneous firing. These seemingly contradictory results underscore the importance of the temporal aspect of nicotine's effects. Mice used for both methods of neuronal activity measurement were sacrificed immediately following the last vapor session. However, spontaneous firing was measured following slice preparation and at least 1-h-long incubation where neurons were potentially in cellular withdrawal with variable levels of nicotine remaining in the brain. Whereas cFos expression reflects a snapshot of neuronal activity ∼30–60 min before the time of sacrifice (Chaudhuri et al., 2000) while the animals are actively being exposed to nicotine vapor. Additionally, the increased cFos following acute and dampened cFos expression following chronic nicotine vapor exposure is similar to what was observed previously in a study examining the patterns of activity following systemic nicotine exposure (Baur et al., 2018) and is potentially regulated by desensitization of nAChRs. This is also consistent with the idea of tolerance where the initial effects and responses to a drug diminish with repeated administration of the drug over time as homeostatic changes develop that oppose the effects of the initial response and potentially more drug is required to achieve effects similar to initial exposure. Phasic GABAergic inhibitory inputs are transiently increased but quickly desensitized leading to an overall increase in excitability in VTA dopamine neurons (Mansvelder and McGehee, 2000; Mansvelder et al., 2002). Although we demonstrate that VTA CRF1 neurons are primarily dopaminergic, phasic inhibitory signaling in VTA-NAcCRF1 neurons was not sensitive to either focal or in vivo nicotine, in contrast to previous work in VTA dopamine neurons. This difference may be because of differences in cell types (dopaminergic vs GABAergic or glutamatergic), species (rat vs mouse), sex (male vs both sexes), and/or age (adolescent vs adult). However, our data suggest that the VTA-NAcCRF1 population displays differential responses to nicotine exposure compared with the VTA dopaminergic population. These differences could underly potentially distinct roles in stress/anxiety and/or addiction.

VTA dopamine neurons are modulated by both phasic (Mansvelder et al., 2002; Grieder et al., 2014) and tonic (Darnieder et al., 2019; Tossell et al., 2021) inhibition. Canonically, phasic inhibition mediates rapid point-to-point signaling while tonic inhibition regulates network activity by persistent inhibitory conductance (Semyanov et al., 2004). In GABAergic VTA neurons, inhibitory tonic currents have been found to be mediated through the δ-containing GABAA receptor with higher receptor mRNA expression and tonic current in female mice and act to disinhibit VTA dopamine neurons (Darnieder et al., 2019). In dopaminergic VTA neurons, inhibitory tonic currents are mediated through αβε-containing GABAA receptors and directly reduce the excitability of those neurons (Tossell et al., 2021). Although inhibitory tonic current has been observed in the VTA, how nicotine impacts this tonic has previously been understudied. In this study, we observed tonic inhibition induced by focal nicotine in naive female and male VTA-NAcCRF1 neurons and this tonic current was enhanced in females and reduced in males following acute nicotine vapor exposure. We speculate that the enhanced tonic observed in females following acute nicotine vapor exposure is potentially because of increased expression of (δ-containing or αβε-containing) GABAA receptors at the extrasynaptic membrane. Concurrently, the sex difference may also be mediated via differential expression or desensitization of nicotinic acetylcholine receptors (nAChRs) on VTA dopaminergic neurons or upstream on GABAergic neurons to alter GABA release and/or reuptake. The enhanced tonic inhibition in females could potentially decrease reward signaling by dampening glutamatergic signaling whereas the reduced tonic inhibition in males could enhance glutamatergic signaling and drive reward signaling. This hypothesis may potentially provide the mechanistic basis for sex differences in motivation for nicotine seeking where women are more likely to seek nicotine to relieve stress and men are more likely to seek nicotine for its rewarding properties. Additionally, these findings suggest that nicotine's effect on excitability in the VTA-NAcCRF1 population may be modulated more by tonic inhibition than phasic inhibition as previously observed in VTA dopamine neurons (Mansvelder et al., 2002; Grieder et al., 2014). Interestingly, the bidirectional sex difference in focal nicotine-induced tonic inhibition was no longer observed following chronic nicotine vapor, suggesting neuroplastic adaptations such as nACh receptor desensitization, internalization, or even decreased gene expression, given the prolonged timeframe, that can diminish the effect of focal nicotine application and dampen the ability of tonic inhibition to regulate VTA-NAcCRF1 activity after chronic exposure. Distinct changes following acute but not repeated nicotine vapor exposure have been observed in our previous work in the central amygdala (Zhu et al., 2021). Additionally, these dampened effects following chronic exposure may be interesting to investigate following withdrawal from nicotine. Overall, these differential effects of nicotine on tonic inhibition, and thus, the excitability of VTA-NAcCRF1 neurons may play a role in the modulation of the reward circuit and the development and maintenance of addiction.

The CRF/CRF1 system is involved in reward processing and has been implicated in many different drugs of abuse. In the context of nicotine, studies report increased CRF mRNA in the VTA following chronic nicotine exposure (Grieder et al., 2014) and CRF1 blockade reduced reinstatement of nicotine seeking (Bruijnzeel et al., 2009). Activation of VTA dopamine CRF1 neurons at their cell bodies has been shown to coordinate reward reinforcement behavior and activation at terminals in the NAc core increase dopamine release (Heymann et al., 2020). Additionally, CRF peptide increases VTA dopamine neuron firing via CRF1 (Wanat et al., 2008; Zalachoras et al., 2022) and knock-out of CRF1 in midbrain dopamine neurons has been shown to increase anxiety-like behavior (Refojo et al., 2011). Previous studies have found Crf1 mRNA expression in GABAergic neurons in the VTA (Zalachoras et al., 2022), however the location of the protein expression (dendrites/cell body vs terminals) and their role in stress/anxiety and nicotine addiction remain unclear. We found that CRF1 VTA neurons were mainly dopaminergic and project to the nucleus accumbens, consistent with previous reports (Heymann et al., 2020); however, VTA-NAcCRF1 neurons did not respond to nicotine in the same way as canonical VTA dopamine neurons. Given the relevance of CRF1 in stress signaling, this suggests that these neurons may be uniquely modulated in the context of stress and anxiety. Future studies examining how nicotine reward processing is affected by stress and anxiety and how anxiety-like behaviors are affected by nicotine exposure are required to understand the intersection of divergent (or convergent) processing in CRF1 VTA neurons in different behavioral conditions.

Collectively, these studies illustrate how CRF1 neurons, which are relevant to stress, are also involved in encoding nicotine reward and how acute and chronic electronic nicotine vapor exposure produce different effects on activity and inhibitory control of VTA CRF1 neurons in females and males. These sex-dependent and exposure-dependent changes in the mesolimbic CRF1 population add to our understanding of the neurobiological underpinnings involved in the development of nicotine addiction. It is imperative that we continue to study how nicotine vapor affects the brain and engages specific reward pathways to better understand nicotine dependence and aid in the development of therapeutics to prevent and mitigate nicotine addiction.

Footnotes

  • This work was supported by NIH Grants F31-DA-053064 (to M.Z.), T32-NS-007431 (to M.Z.), AA-026858 (to M.A.H.), and AA-011605 (to M.A.H.). We thank Dr. Charles R. Esther and Tara Nicole Guhr Lee for their help with the serum nicotine and cotinine analysis. We also thank Maria Echeveste Sanchez for technical assistance.

  • The authors declare no competing financial interests.

  • Correspondence should be addressed to Melissa A. Herman at melissa_herman{at}unc.edu

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Sex Differences in the Impact of Electronic Nicotine Vapor on Corticotropin-Releasing Factor Receptor 1 Neurons in the Mouse Ventral Tegmental Area
ManHua Zhu, Neil G. Rogers, Jasmine V. Jahad, Melissa A. Herman
Journal of Neuroscience 26 April 2023, 43 (17) 3081-3093; DOI: 10.1523/JNEUROSCI.2087-22.2023
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