Paternal nicotine enhances fear memory, reduces nicotine administration, and alters hippocampal genetic and neural function in offspring

Nicotine use remains highly prevalent with tobacco and e‐cigarette products consumed worldwide. However, increasing evidence of transgenerational epigenetic inheritance suggests that nicotine use may alter behavior and neurobiology in subsequent generations. We tested the effects of chronic paternal nicotine exposure in C57BL6/J mice on fear conditioning in F1 and F2 offspring, as well as conditioned fear extinction and spontaneous recovery, nicotine self‐administration, hippocampal cholinergic functioning, RNA expression, and DNA methylation in F1 offspring. Paternal nicotine exposure was associated with enhanced contextual and cued fear conditioning and spontaneous recovery of extinguished fear memories. Further, nicotine reinforcement was reduced in nicotine‐sired mice, as assessed in a self‐administration paradigm. These behavioral phenotypes were coupled with altered response to nicotine, upregulated hippocampal nicotinic acetylcholine receptor binding, reduced evoked hippocampal cholinergic currents, and altered methylation and expression of hippocampal genes related to neural development and plasticity. Gene expression analysis suggests multigenerational effects on broader gene networks potentially involved in neuroplasticity and mental disorders. The changes in fear conditioning similarly suggest phenotypes analogous to anxiety disorders similar to post‐traumatic stress.


| INTRODUCTION
Accumulating evidence suggests that the impact of exposure to drugs of abuse extends beyond the individual to affect physiological and behavioral phenotypes in unexposed offspring. [1][2][3] Characterization of nicotine's effects across generations is critical considering the prevalence of tobacco product use 4 and the dramatic rise of e-cigarette use. 5 Through its effects on brain cholinergic systems, nicotine exposure produces marked alterations in brain function that may underlie nicotine addiction and contribute to increased risk for psychiatric disorders, including depression 6 and anxiety. 7 Epigenetic modifications downstream of cholinergic activation may allow for persistent effects on cellular and circuit function. 8,9 Until recently, it was believed that these epigenetic modifications were erased upon *Lisa R. Goldberg & Dana Zeid contributed equally to the manuscript and are co-first authors. establishment of the germline and thus sequestered from subsequent generations. However, epigenetic modifications, including DNA methylation, histone posttranslational modifications, and noncoding RNAs, acquired in one generation can be inherited in the next generation. 10,11 These epigenetic modifications may mediate multigenerational and transgenerational effects of parental nicotine exposure on offspring behavior and neurobiology.
Rodent studies from multiple, independent laboratories have begun to identify multigenerational and transgenerational consequences of parental nicotine exposure. This work has thus far identified effects of parental nicotine exposure on depressive-and anxiety-like phenotypes, 1 cognitive flexibility, 2 attention deficit hyperactivity disorder (ADHD)-like behaviors, 3 and gene expression. 1,2 The multigenerational and transgenerational consequences of nicotine exposure may affect endophenotypes involved in nicotine addiction and mental health. For example, we have shown that nicotine exposure modulates contextual fear conditioning, a model of hippocampus-dependent fear learning that is related to vulnerability to mental health disorders such as post-traumatic stress disorder (PTSD) and addiction. [12][13][14] Nicotine's effects on contextual fear learning are modulated by the hippocampus. 15, 16 We have found that acute nicotine exposure enhances hippocampus-dependent fear learning, 15,17 impairs extinction of contextual fear, 18,19 and augments spontaneous recovery of contextual fear following extinction. 18 However, the multigenerational and transgenerational effects of paternal nicotine exposure on these phenotypes have not been studied.
Furthermore, no previous studies have characterized nicotine's multigenerational effects on cholinergic function. Multigenerational inheritance refers to phenotypes arising in the generation immediately following exposed individuals, whereas transgenerational inheritance consists of germ-line-mediated inheritance of epigenetic information between generations in the absence of direct environmental influences that lead to phenotypic variation. 11 Here, we examined both multigenerational and transgenerational effects of paternal nicotine exposure on contextual and cued fear learning in the F1 and F2 generation as well as on nicotine self-administration, hippocampal nicotinic acetylcholine receptor (nAChR) binding, hippocampal cholinergic functioning, hippocampal gene expression, and hippocampal DNA methylation in the F1 generation. We hypothesize that paternal nicotine exposure will impact fear conditioning, hippocampal gene expression, and function in offspring and grand-offspring.

| Subjects
Subjects were male and female C57BL/6J mice (8-20 weeks of age, Jackson Laboratory, Bar Harbor, ME). With the exception of housing for harem breeding, all animals were group-housed with a 12-hourr light/dark cycle and ad libitum access to food and water. During self-administration, subjects were food-restricted to 85% to 90% of their free-feeding body weight and water was provided ad libitum.
All behavioral testing occurred between 9:00 AM and 6:00 PM. All procedures were conducted in accordance with the NIH Guide for the

| Generation of F1 and F2 mice
The half-life of nicotine in mice is approximately 6 minutes. 22 It has been shown previously that the impacts of nicotine withdrawal dissipate by 4 days post-nicotine removal. [23][24][25] Therefore, a 4-day delay between nicotine treatment and breeding was implemented to ensure systemic elimination of nicotine prior to breeding. Male mice were placed into cages with two naïve C57BL/6 J females (8-20 weeks of age) for 2 weeks to generate F1 offspring. F2 mice were generated by mating naïve male F1 mice with naïve females.

| Fear conditioning
Fear conditioning and extinction procedures have been described in detail previously. 19 Briefly, mice were trained and tested in noise-attenuating chambers ( 19 To examine potential ceiling effects during cued testing, a separate cohort of F1 mice received identical training with only one CS-US pairing, and contextual fear extinction and spontaneous recovery were also examined. Fear extinction occurred over five consecutive sessions beginning the day after contextual and cued fear testing. Following the final extinction session, the mice were left undisturbed in their home cages for 7 days and then retested in the training context for spontaneous recovery. To determine if any observed differences in fear conditioning were due to differences in shock sensitivity, anxiety, or more broad learning deficits, male and female NIC-and SAL-Sired animals were additionally tested in open field, shock sensitivity, elevated plus maze (EPM), and novel object recognition paradigms (see Supporting Information for full methods and results).

| Food and intravenous nicotine self-administration
A separate cohort of adult SAL-Sired and NIC-Sired F1 mice were used for food and nicotine self-administration studies. Beginning at 6 weeks of age, male F1 mice were weighed, mildly food-restricted to 85% to 90% of their free-feeding body weight, and then trained to press a lever in an operant chamber (Med Associates) for food chow pellets (20 mg; TestDiet, Richmond, IN) under a fixed-ratio 5, time out 20 seconds (FR5TO20 sec) schedule of reinforcement (see Supporting Information for full methods). Once stable responding was achieved (>25 pellets per session across three subsequent sessions), subjects were jugular vein catheterized under isoflurane (1%-3%)/oxygen vapor anesthesia, as previously described. 26 Mice were allowed greater than or equal to 72 hours to recover from surgery before access to respond again for food reward. The re-establishment of food responding ensures that the mice have sufficiently recovered post-intravenous surgery and exhibit normal operant responding following a delay in access to the operant chambers. The mice were then permitted to acquire intravenous (IV) nicotine self-administration during 1-hour daily sessions, 6 to 7 days per week (nicotine hydrogen tartrate salt dissolved in 0.9% sterile saline, 0.03 mg/kg/infusion, free base weight; MP Biomedical, Santa Ana, CA). IV nicotine was delivered by a Razel syringe pump (Med Associates). Each session had two retractable levers (one active, one inactive). Completion of the response criteria on the active lever resulted in the delivery of an IV nicotine infusion (0.03 ml infusion volume; FR5TO20 sec schedule). Responses on the inactive lever were recorded but had no scheduled consequences. Following eight acquisition sessions at 0.03 mg/kg/infusion, the infusion dose switched to 0.1 mg/kg/infusion for six sessions. For each dose, mean intake of the last three sessions was used for statistical analyses. Catheters were flushed daily with physiological sterile saline (0.9% w/v) containing heparin (100 USP units/ml). Catheter patency was verified with Brevital (methohexital sodium, Eli Lilly, Indianapolis, IN) following the nicotine selfadministration phase. To assess relapse-related behavior, the mice were tested for incubation of craving after the session immediately following the last 0.1 mg/kg/infusion dose of IV nicotine selfadministration; in this procedure, the mice are permitted to respond on the active lever but receive no infusions of nicotine. On the first baseline incubation session (day 1), mice were placed in operant chambers under the FR5TO20 sec schedule with contingent cue light activation. Thereafter, the mice were housed in home cages for 20 days.
On day 21 of abstinence, the mice were examined for incubation of craving, with the active lever cue light being delivered under the FR5TO20 sec schedule. Studies were conducted by experimenters blinded to group conditions, and behavioral responses were automatically recorded by MedAssociates software.

| Nicotinic acetylcholine receptor binding
A radioligand binding assay 16   Ventral and dorsal hippocampus were evaluated separately, as they differentially contribute to contextual fear conditioning: the ventral hippocampus (vHPC) has a more prominent role in fear association and expression, while the dorsal hippocampus (dHPC) is critical for contextual memory. 31 Ag/AgCl reference electrodes were implanted into contralateral rostral cortex.
Amperometric recordings were conducted at 2 Hz by applying a fixed potential of +0.7 V, and data were digitized (FAST-16 potentiostat, Quanteon, Nicholasville, KY). Background currents were stabilized for 60 minutes, then drugs were applied into the hippocampus using a glass capillary (tip diameter:15 μm) attached to the electrode. Depolarization-evoked ACh release was measured by applying either brief pulses of potassium (KCl 70 mM; 100 nL) or NIC (1 mM freebase, nicotine tartrate; 100 nL) at 2 to 10 psi every 2 minutes.
Recordings were counterbalanced for hippocampal region (dorsal or ventral) and drug (potassium or NIC). Choline signal amplitudes were measured by change in current on enzyme-coated channel from baseline current and converted into μM equivalents of choline based on in vitro calibration. Self-referencing was adopted to eliminate artifacts by subtracting currents from sentinel channels. 29 Microelectrode placement was verified by Nissl staining of coronal hippocampal sections ( Figure S1). Averages of two responses per drug manipulation per animal were used for statistical analysis.    Table S1). 38  were removed and low-quality bases were trimmed using Trimmomatic. 39 Low-quality base trimming was performed with a sliding window approach, trimming when the average quality within a four base pair window fell below a threshold of 20, with a minimum read length of 35. After trimming, FASTQ files possessed mean per read Phred quality scores greater than 30 (ie, less than 0.1% sequencing error). Trimmed reads were mapped to the mouse reference genome (mm10) using Bowtie2 40 implemented in Bismark. 41 Methyl_extract within Bismark was used to extract CpG methylation information and to create methylation reports. MethylKit 42 was used for analysis of differentially methylated regions (DMRs). Methylation status was summarized over nonoverlapping windows of 500 base pairs and differential methylation analysis was performed, with a standard FDR of 0.05. 35 Datasets have been deposited to Gene Expression Omnibus.

| RESULTS
3.1 | Paternal nicotine enhances contextual fear conditioning and reverses acute nicotine enhancement of contextual fear conditioning in F1 and F2 generation mice NIC-Sired and SAL-Sired F1 male and female mice were fear conditioned following acute SAL or NIC administration (0.09 mg/kg i.p., Figure 1A). Complete analysis of baseline, pre-CS, and CS freezing is included in Supporting Information. A 3-way ANOVA of contextual freezing with sire treatment, acute drug treatment, and sex as factors revealed a significant sire × acute drug treatment interaction (F (1,36) = 32.75, P < .001). Because there was no significant interaction between sex and sire or acute drug treatment, a 2-way ANOVA collapsed across sex was performed and revealed a significant sire treatment × acute drug treatment interaction (F (1,40) = 20.96, P < .001). Post hoc comparisons indicated that saline-treated NIC-Sired F1 mice exhibited augmented contextual fear conditioning compared with saline-treated SAL-Sired F1 mice (t 20 = 2.73, P < .05).
Overall, context freezing levels in NIC-Sired NIC mice were comparable with those observed in SAL-Sired SAL mice at both 0.09 mg/kg (P > .05).
Additionally, male and female NIC-and SAL-Sired animals were tested in shock sensitivity ( Figure S2), elevated plus maze (EPM, Figure S3), open field, and novel object recognition paradigms ( Figure S4, see Supporting Information for full methods and results).
With the exception of NIC-Sired females in EPM (who showed increased anxiety-like behaviors) and NIC-Sired animals in shock sensitivity (who showed decreased vocal reactivity to shock, which would not confound the enhanced fear learning multigenerational phenotype), no differences were detected between NIC-and SAL-Sired mice.

FIGURE 1
Paternal nicotine enhances contextual fear conditioning and attenuates acute nicotine enhancement of fear conditioning. A, Contextual freezing was significantly higher in NIC-Sired + SAL compared with SAL-Sired + SAL controls. Acute nicotine at 0.09 mg/kg enhanced contextual fear conditioning in SAL-Sired but significantly reduced contextual fear conditioning in NIC-Sired animals (n = 10-12 per group). B, Contextual freezing was significantly higher in NIC-grandsired + SAL compared with SAL-grandsired + SAL controls (n = 9-11 per group). Error bars indicate standard error of the mean (SEM), * P < .05 To determine if impaired contextual fear conditioning continued into the next generation (F2), NIC-grandsired and SAL-grandsired male and female F2 mice were bred from naïve F1 male mice.
Complete analysis of baseline, pre-CS, and CS freezing is included in Supporting Information. A 3-way ANOVA of contextual freezing was performed with grandsire treatment, acute drug treatment, and sex as independent factors ( Figure 1B). Because there was no significant interaction between sex and sire or acute drug treatment, a 2-way ANOVA collapsed across sex was performed. A significant main effect of sire (F (1,37) = 9.88, P < .01) was found, and post hoc comparisons indicated that NIC-grandsired mice exhibited augmented contextual fear conditioning compared with SAL-grandsired mice (t 39 = 3.04, P < 0.01). In addition, SAL-grandsired mice administered acute NIC had enhanced contextual fear conditioning (t 19 = 2.41, P = 0.026) but acute NIC did not enhance contextual fear conditioning in NIC-grandsired mice.

| Paternal nicotine enhances cued fear conditioning in F1 generation mice
To examine whether a ceiling effect precluded detection of group differences for cued fear conditioning (see Supporting Information), a separate group of F1 mice was trained with one CS-US pairing. In this cohort, enhancement of contextual fear conditioning (t 7 = 3.21, P < .05) as well as cued fear conditioning was found in NIC-Sired mice (t 6 = 2.41, P < .05; Figure 2A).

| Paternal nicotine enhances spontaneous recovery of contextual fear memory in F1 generation mice
The cohort of F1 mice that received one CS-US pairing were subsequently tested for extinction and spontaneous recovery of contextual fear memory. F1 NIC-Sired mice showed normal fear extinction but displayed enhanced spontaneous recovery of contextual fear memory relative to SAL-Sired mice (t 7 = 3.38, P < 0.05; Figure 2B).

| Paternal nicotine decreases nicotine self-administration
Prior to training for nicotine self-administration, the subjects were analyzed for their ability to learn an operant task to obtain food reward and no differences were observed (Supporting information, Figure S5). To test potential effects of sire nicotine exposure on nicotine reinforcement in F1 offspring, acquisition of IV nicotine self-administration (0.03 mg/kg/infusion) was assessed in a 2-way mixed design ANOVA, which identified a main effect of session (F (7,119) = 13.60, P < .001) and a session × sire treatment interaction (F (7,119) = 5.00, P < .001). However, post hoc tests did not reveal any statistically significant differences between the groups on each of the eight acquisition sessions ( Figure 3A). The number of active and inactive lever presses were then analyzed to determine if the groups maintained an across-session preference for the active lever during acquisition ( Figure 3B), which identified a main effect of session (F (7,238) = 24.18, P < .001) and a session × sire treatment interaction (F (21,238) = 11.40, P < .001). Post hoc analysis revealed that the groups differed on the first day of nicotine self-administration. The NIC-Sired group exhibited greater active lever pressing compared with the SAL-Sired group. This effect may either represent a greater level of drug-seeking behavior on the first day of exposure, perseverance of responding for food reward, and/or decreased cognitive flexibility in transitioning responding from food to drug. However, this difference did not persist across further sessions. SAL-Sired mice exhibited a consistent statistically significant preference for the active lever over their inactive lever (post hoc P < .01), but NIC-Sired mice did not exhibit this maintained preference for sessions 3 to 8.
To further examine potential group differences while controlling for variability during the initial phase of acquisition, the mean number of nicotine infusions were examined for the last three sessions, a time at which the subjects displayed more consistent responding for nicotine ( Figure 3C). The groups did not significantly differ in the mean number of nicotine infusions (P > .05). Thereafter, the mice were transitioned to a 0.1 mg/kg/infusion dose of nicotine, previously shown to be preferred in adult C57BL6/J mice. 44 At this dose,  Figure 3D). For incubation of craving behavior, which is considered a measure of increased drug seeking during abstinence, a 2-way mixed design ANOVA with session and sire treatment identified a main effect of session (F (1,17) = 16.90, P < .001). While SAL-Sired animals exhibited an incubation effect with greater responding on day 21 of abstinence as compared with day 1, NIC-Sired mice did not display an increase in nicotine seeking behavior (P < .01).
Amperometric recordings of potassium-and nicotine-evoked ACh currents were assessed in F1 dHPC and vHPC. Due to uneven sample sizes per sex, sex was not included as a preliminary factor in these analyses. KCl depolarization-evoked cholinergic signals did not differ between SAL-and NIC-Sired mice in dHPC (P > .05; Figure 4A); however, local nicotine application resulted in a significant reduction in cholinergic signal amplitudes in NIC-Sired mice (t 8 = 2.33, P < .05; Figure 4C). In vHPC, ACh release was decreased in NIC-Sired mice following application of KCl (t 8 = 2.60, P < .05; Figure 4B) or nicotine (t 8 = 2.98, P < .05; Figure 4D).  Table S2).

| Paternal nicotine exposure differentially alters dorsal and ventral hippocampal gene expression
Of these genes, 612 were downregulated and 340 were upregulated in NIC-Sired mice. In dHPC, only 162 genes were differentially expressed in NIC-Sired mice compared with SAL-Sired mice (FDR = 0.05). Of these 162 genes, 86 were downregulated and 76 were upregulated. One hundred three genes with altered gene expression overlapped between vHPC and dHPC.  Paternal nicotine reduces nicotine self-administration. A, NIC-and SAL-Sired male mice (n = 9-10 per group) did not differ in the total number of infusions earned for each session during the acquisition period on the 0.03 mg/kg/infusion dose. B, During acquisition, the number of active and inactive lever presses significantly differed on the first session, with NIC-Sired mice nicotine exhibiting a greater number of active lever presses compared with SAL-Sired mice. However, across subsequent sessions, NIC-Sired mice decreased responding, resulting in no significant differences between their active and inactive number of lever presses across sessions 3 to 8. In contrast, SAL-Sired animals exhibited a consistent statistically significant preference for the active lever over their inactive lever. C, Mean number of nicotine infusions across the three last acquisition sessions did not significantly differ between NIC-and SAL-Sired mice. D, At a moderate dose of 0.1 mg/kg/infusion, NIC-Sired mice selfadministered a significantly lower number of nicotine infusion. E, Incubation of craving assessment revealed a significant increase in responding on the previously active lever after 21 days of abstinence only for SAL-Sired mice. Error bars indicate standard error of the mean (SEM), * P < .05
Complementary to the IPA results, enrichment analysis using Enrichr provided further evidence for alterations in cellular growth and development in vHPC, with top GO biological terms including "RNA splicing," "response to unfolded protein," and "regulation of cell growth" and "protein stabilization" (Table S5)
Differentially expressed genes unique to dHPC and vHPC were subsequently analyzed separately in IPA in order to test for divergent neurobiological adaptions between the two regions (Table S6)  conditioning. 16,21 In the present study, acute nicotine enhanced contextual fear conditioning in F1 and F2 mice from saline-treated mice.
In contrast, acute nicotine-disrupted contextual fear conditioning in NIC-Sired mice and had no effect in NIC-grandsired mice, which may point to altered cholinergic functioning in the hippocampus.
The effects of paternal nicotine exposure on subsequent nicotine self-administration in the F1 generation also points to disrupted cholinergic function. During acquisition of IV nicotine self-administration at the lower dose, the groups did not differ in the number of nicotine infusions, although an increase in the number of active lever presses was found in the NIC-Sired group. This suggests that the NIC-Sired mice may have exhibited a perseverance of responding for food reward and/or decreased cognitive flexibility in transitioning responding from food to drug. However, it is also worthwhile to note that the groups did not differ on day 1 of incubation of craving, which represents an extinction session (eg, no nicotine infusions during session), and thus, this effect appears to have been present when reinforcers are switched but not in the absence of a reinforcer during an extinction session. The NIC-Sired mice also exhibited decreased nicotine self-administration at the moderate dose, which aligns with recent work identifying decreases in alcohol, cocaine, and opioid administration associated with parental alcohol, cocaine, and morphine exposure (eg Vassoler et al, 46 as reviewed in Goldberg and Gould 47 ). The observed reduction in nicotine self-administration may be attributed to either decreased sensitivity to the rewarding effects of nicotine and/or increased sensitivity to the aversive effects of nicotine. Indeed, the groups differed at the moderate nicotine dose but not the lower nicotine dose, which supports the notion of an increased aversive response with the higher dose. Interestingly, we also found a lack of incubation of craving on day 21 in NIC-Sired mice following self-administration at the moderate dose, suggesting that decreased nicotine-seeking behaviors could be related to an aversion-associated memory for nicotine. Although various neural substrates may underlie these effects on nicotine intake and relapse-related responding, a recent study found that decreased DNA methyltransferase in the hippocampal CA1 region reduced morphine self-administration. 48  DHPC is known to modulate contextual fear conditioning. 31,50 Inhibition of vHPC disrupts both cued and contextual fear conditioning 51,52 and vHPC cholinergic lesions impair cued fear conditioning. 53 We have also shown that direct nicotine infusion into dHPC enhances contextual fear conditioning while infusion into vHPC disrupts contextual fear conditioning. 15 57 where mice were exposed to an electric footshock and then presented situational reminders. Fkbp5 encodes a glucocorticoid receptor chaperone whose functioning has been associated with a maladaptive prolonged stress response in individuals with PTSD and other anxiety disorders. 58 Specifically, human studies show that Fkbp5 methylation and transcription correlate with severity of PTSD symptoms, such that increased methylation and decreased transcription predict more severe PTSD symptomology. 59,60 Fkbp5 expression modulates HPA-axis functioning, which is thought to mediate its involvement in PTSD. 59,61 Our finding of enhanced spontaneous recovery of fear memory in conjunction with dysregulation of transcriptional pathways associated with glucocorticoid signaling in NIC-Sired animals may point to increased vulnerability to PTSD-like phenotypes.
In dHPC, DMR patterns were largely inconsistent with the direction of differential transcript expression found by RNA-sequencing, which suggests that changes in vHPC DNA methylation produced by paternal nicotine exposure are more consequential in terms of impacting gene expression than those in dHPC. This is in line with our identification of a greater number of differentially expressed transcripts and more exaggerated changes in cholinergic transmission in NIC-Sired vHPC compared with dHPC. Our targeted sequencing approach may have limited the ability to detect potential transcriptional regulation by distally methylated sequences. Future investigations including analysis of genome-wide DNA methylation, histone modifications, and small RNA expression will provide a more complete interpretation of these findings.
A potential limitation of our nicotine exposure design is the focus on paternal nicotine exposure to investigate the multigenerational and transgenerational impact of nicotine exposure. Although other studies finding multi/transgenerational phenotypes following paternal drug exposure, including cocaine 46 and morphine, 62 found no differences in maternal care, it is possible that paternal nicotine exposure may impact maternal care. Future studies investigating the impact on maternal care are warranted. As our current focus was on paternal exposure, future work should also compare impacts of paternal vs maternal exposure.
Overall, the present findings provide a novel understanding for the multigenerational and transgenerational effects of nicotine exposure, which are supported by a growing literature characterizing multigenerational and transgenerational effects of drug exposure (as reviewed in Goldberg and Gould 47 ). This study was the first to test contextual fear conditioning in F1 and F2 offspring of nicotine-exposed males and identified enhanced fear memory formation and spontaneous recovery of fear memories. This study was also the first to identify altered nicotine self-administration and incubation of craving in F1 nicotineexposed offspring. Differential methylation in genes associated with PTSD and HPA-axis dysregulation as well as concurrent disruptions in stress-related transcriptional pathways were found in NIC-Sired mice. Paternal nicotine was also associated with decreased hippocampal cholinergic function and increased hippocampal nAChR binding.
Interestingly, PTSD patients that did not smoke show significantly higher mesiotemporal cortical high-affinity nAChR binding 63