The proposed study evaluated sex differences in glutamate (Glu), with a focus on the dorsal anterior cingulate cortex (dACC), anterior insula, and thalamus, as well as how it is influenced by sex (males vs. females), smoking state (overnight abstinent vs. sated), and circulating ovarian hormones (estrogen and progesterone) in women. Glu was measured in the entire brain with special focus on the dorsal anterior cingulate cortex (dACC), anterior insula, and thalamus, all of which have been implicated in tobacco withdrawal, using an echo-planar spectroscopic imaging (EPSI) variant of magnetic resonance spectroscopy (MRS). Serum ovarian hormones (estrogen and progesterone) were measured for female participants to determine relationships between brain Glu and this hormone. Glu was be measured in smokers after overnight (~12 h) abstinence and after participants smoked the first cigarette of the day.

Glutamatergic signaling is dysregulated in addictions, including Tobacco Use Disorder (TUD) , and represents a promising target for smoking cessation therapies. In rodents, NMDA-type glutamate (Glu) receptor antagonists reduce nicotine-seeking behavior, whereas pre-treatment with NMDA-type and AMPA-type Glu receptor antagonists attenuates nicotine self-administration and nicotine-induced dopamine release. In humans, N-acetylcysteine, which regulates Glu via the cysteine-glutamate antiporter and glial Glu transporter, reduces cigarette smoking9 as well as cortical and subcortical levels of Glu itself, measured by magnetic resonance spectroscopy (MRS). These observations suggest that Glu, assayed by MRS, which is a safe and non-invasive in vivo metric, may aid in tracking regional effects of smoking and of anti-smoking interventions.

Notably, evidence has been provided that Glu in the dorsal anterior cingulate cortex (dACC) is associated with smoking-related states, that Glx (the sum of Glu and its primary metabolite, glutamine, Gln) in dACC is higher in smokers than nonsmokers, and that Glx in the insula is higher after overnight abstinence from smoking than during satiety. Further, studies in smokers combining functional magnetic resonance imaging (fMRI) with MRS have linked dACC Glu with activation of the Default Mode Network during cue-induced cigarette craving, and demonstrated reduced dACC Glx and altered dACC-to-DMN connectivity following varenicline treatment. Prior MRS studies of smoking, however, have been limited by single-voxel techniques that sample single brain regions with restricted spatial-resolution and partial volume effects (i.e., including more than one tissue type in the acquisition voxel).

We will exploit the echo-planar spectroscopic imaging (EPSI) variant of MRS in a high-density, anatomically broad assessment of how sex, acute smoking, and ovarian hormones affect brain Glu. Our pilot data indicate that dACC Glu is lower in smoking-abstinent women than men, and lower still in women with higher serum estrogen. After smoking, dACC Glu decreases in men but not in women.

We will use EPSI to determine whether our dACC Glu pilot findings: 1) replicate at high spatial resolution; 2) pertain to the anterior insula and thalamus, which are also implicated in TUD, and 3) on an exploratory basis, pertain to other regions in the rest of the brain. To accomplish this, we will use a recently developed short echo-time (TE=20 ms) variant of 3D EPSI at 3-Tesla. With this state-of-the-art technique, we typically acquire high-quality spectra simultaneously from ~80% of the brain at the very high spatial resolution of ~0.4 cc. Short-TE, moreover, improves segregation of Glu from the overlapping Gln signal.

We teste adult daily smokers (men and women) before and after their first cigarette of the day after ~12 h abstinence. Serum estrogen and progesterone in women were assayed. We addressed two specific aims:

Aim 1: Determine relationships between brain Glu, sex, and circulating ovarian hormones. Hypothesis 1a: Our single-voxel MRS finding that men have higher Glu in the dACC than women after overnight abstinence from smoking will be replicated with EPSI, and will extend to the anterior insula and thalamus. Hypothesis 1b: EPSI will replicate our preliminary finding that Glu in the dACC correlates negatively with serum estrogen (and possibly progesterone) in women, and will show similar relationships of ovarian hormones with the anterior insula and thalamus.

Aim 2: Determine sex differences in acute effects of smoking on brain Glu. Hypothesis 2: The preliminary finding that Glu decreases after acute smoking in the dACC of men but increases or is unchanged in women will be replicated, and will extend to other brain regions.

Ultimately, measurements of sex differences in Glu in specific brain regions, and how Glu changes with smoking and ovarian hormones, can define biomarkers that facilitate development of novel treatments and evaluate therapeutic response in men and women.

Inclusion Criteria:

1. Self-identified as only male or female
2. Age 18-45 years (children <18 years will be excluded due to low prevalence of conventional cigarette smoking; female participants >45 years of age will be excluded to avoid effects of perimenopause and menopause in women; male participants >45 years of age will be excluded as well to ensure that male and female groups are matched on age
3. English fluency demonstrated by verbal skills sufficient to participate in a conversation, including the ability to ask and answer questions at a level that assures adequate understanding of the study (a comprehension quiz will be given)
4. Right handedness (evaluated using the Edinburgh Inventory)
5. Generally in good health without cardiovascular, hepatic, renal, or autoimmune diseases, diabetes, or cancer
6. Must have smoked for ≥1 year
7. Must endorse inhaling while smoking
8. Must smoke ≥10 cigarettes per day
9. Must have expired CO >10 ppm and urinary cotinine ≥100 ng/ml at screening/intake
10. Fulfillment of DSM-5 criteria for Tobacco Use Disorder

Exclusion Criteria:

1. Seeking treatment for nicotine dependence within 3 months of screening
2. Medical condition that may compromise safety (based on history, physical exam)
3. Neurological disorder that would compromise compliance and/or informed consent
4. Major psychiatric disorder (e.g., Major Depression, Schizophrenia, Bipolar Disorder) per DSM-5 MINI
5. Current drug use disorders other than Tobacco Use Disorder (as defined in DSM-5)
6. Recent use of cocaine, opiates, benzodiazepines, or amphetamines as shown by urine test at the screening or testing sessions
7. Smoke marijuana >3X/week (self-report) or positive marijuana urine test on a scan day (positive at screening allowed)
8. Use of tobacco in forms other than cigarettes (e.g., snuff, chewing tobacco, e-cigarettes) >10 days in the month before screening
9. Preference for menthol cigarettes, given sex differences in the effect of menthol on the rate of nicotine entry into the brain
10. Pregnancy or nursing
11. Presence in the body of metal that would compromise safety during MRI
12. Claustrophobia
13. Any other condition that would compromise safety

Target recruitment: 60 adult daily smokers (30 men and 30 women; 18-45 years old), who are otherwise in good health. Interested individuals will be invited to participate in the research project regardless of sex, race or ethnicity, although strong efforts will be made to recruit equal numbers of male and female participants. Individuals will not be excluded from study participation based on race/ethnicity.

The primary assessment is a sex difference in neurometabolites in brain measured using MRS. This was an observational study. There was no pharmacological intervention. Participants were tested after overnight abstinence form smoking -- before and after they smoked the first cigarette of the day.

Adverse event data were collected on the study day.

Description of adverse events does not differ from the clinicaltirals.gov Definitions.