Smoking is highly prevalent among patients with serious mental illness and this co-occurrence leads to medical and psychiatric morbidity (Green et al., 1999, 2008; Mallet et al., 2019) as well as an increased mortality risk (Tran et al., 2009; McGinty et al., 2012; Dickerson et al., 2018). Specifically, patients with schizophrenia (SCZ) have higher smoking rates than the general population (de Leon and Diaz, 2005) with lifetime prevalence reported at 60–90% (Volkow, 2009). In one investigation studying patients with SCZ or bipolar disorder, current smokers showed worse cognitive functioning and had poorer functional outcomes than past or never smokers. These effects were observed regardless of diagnosis, however, the patients with SCZ were twice as likely to be smokers compared to those with bipolar disorder (Depp et al., 2015). Moreover, in a recent study, 31% of current smokers were readmitted to a psychiatric hospital within 1 year of discharge compared to 26% of never smokers (Kagabo et al., 2019). Collectively, these studies indicate a correlation between smoking in patients with a serious mental illness and increased psychiatric morbidity and mortality.

The underlying causes of co-occurring mental illness and substance use disorders are largely unknown. However, there is evidence indicating that genetic factors combined with prenatal and/or postnatal developmental insults (including the use of drugs during adolescence; Khokhar et al., 2017), contribute to the development of these disorders. A number of studies suggest cannabis use (Fergusson et al., 2003), and tobacco smoking (Gage and Munafo, 2015; Kendler et al., 2015) may be associated with increased psychotic symptoms. Additionally, for many patients substance use precedes psychosis, with reports finding that substance use rates among patients with first episode psychosis are 30–70% (Abdel-Baki et al., 2017). Thus it is important to study substance use during adolescence and its potential role in contributing to an individual’s risk of developing a psychiatric diagnosis.

One developmental insult used in rats that results in several dysregulated behavioral endophenotypes is the neonatal ventral hippocampal lesion (NVHL). NVHL rats display symptoms resembling those occurring across psychiatric disorders, though they are often used as a model of SCZ (Lipska et al., 1993, 1995; Sams-Dodd et al., 1997; Brady et al., 2010; Gruber et al., 2010; Placek et al., 2013). Moreover NVHL rats self-administer drugs, including nicotine, at a higher rate than normal rats (Chambers and Self, 2002; Berg et al., 2011; Sentir et al., 2020), as well as demonstrate enhanced nicotine behavioral sensitization (Berg and Chambers, 2008). Behavioral sensitization is the progressive increase of drug-induced locomotion with repeated exposure to a drug (Robinson and Berridge, 1993) and is a phenomenon documented in both humans and animals (Kalivas and Stewart, 1991; Robinson and Berridge, 2008). This behavior is indicative of neuroadaptations occurring in motivation related brain regions underlying drug-wanting and craving (Robinson and Berridge, 2008) and can be affected by perturbations occurring in adolescence (McCormick et al., 2004; Mathews et al., 2008; McCormick, 2010; Garcia et al., 2017). Furthermore, cross-sensitization has also been shown, where the repeated exposure of one drug yields sensitization to another drug (Kalivas and Stewart, 1991; Steketee and Kalivas, 2011).

Neonatal ventral hippocampal lesion rats have also been shown to increase alcohol consumption in adulthood after voluntary adolescent alcohol intake (Jeanblanc et al., 2015). Alcohol remains one of the most commonly used drugs by adolescents [Johnston et al., 2020; Substance Abuse and Mental Health Services Administration (SAMHSA), 2020], and as such, combining the NVHL developmental insult with adolescent alcohol exposure can be used to study the complex dynamics between adolescent drug use, SCZ, and increased smoking. In the present study, we used the NVHL rat to determine how exposure to adolescent alcohol affects nicotine behavioral sensitization in adulthood. In humans, alcohol use during adolescence has been linked to increased substance use in adulthood (Ellickson et al., 2003; Grant et al., 2006; Ryan et al., 2019), therefore, we hypothesized NVHL animals with alcohol exposure would demonstrate increased nicotine behavioral sensitization.

Here, we sought to determine the effects that alcohol exposure during adolescence would have on nicotine behavioral sensitization in the NVHL model of SCZ. The results suggest that adolescent alcohol exposure from PD 28–42 did not alter the amount of nicotine behavioral sensitization. When controlling for baseline differences in distance traveled, there were no differences in the amount of nicotine behavioral sensitization between NVHL and sham rats, regardless of adolescent alcohol exposure. Importantly though, all groups did sensitize to nicotine, as demonstrated by a significant treatment^∗day interaction in distance traveled and the significant increase in percentage change across the 15 sessions (Figure 5).

The NVHL no AE group showed a significant increase in distance traveled during the preinjection phase of the 15 sessions (Figure 4A), in addition to a significant increase in velocity (Figure 4E). While previous studies have found that postpubertal NVHL rats show spontaneous hyperlocomotion (Lipska et al., 1993; Sams-Dodd et al., 1997), as well as increased locomotor response to a novel environment (Berg and Chambers, 2008), we saw no group differences in distance traveled during the preinjection phase on Day 1. However, we did observe that the NVHL no AE group showed significant increases in distance traveled during the preinjection phase across days once nicotine injections began, suggesting the development of context sensitization, a phenomenon that has previously been reported in the literature in normal rats. Rats treated with nicotine (0.6 mg/kg) for 9 days showed an increase in locomotor activity compared to saline treated animals in the 30 min prior to drug administration (Kosowski and Liljequist, 2005). Similarly, an environment repeatedly paired with nicotine (0.6 mg/kg) acquired the ability to elicit increases in activity even in the absence of nicotine (Walter and Kuschinsky, 1989; Bevins et al., 2001). These data are the first to report that the NVHL rat has enhanced context sensitization, possibly pointing to an increase in the salience of nicotine, and a shift of that salience from nicotine to the context, in this group. Furthermore, it appears that adolescent alcohol exposure impairs the formation of context sensitization, possibly by dampening the salience of nicotine, in the NVHL AE rat. An increase in the salience of nicotine is supported by previous work demonstrating that NVHL rats have increased nicotine seeking behavior during extinction than their sham counterparts (Berg et al., 2013; Rao et al., 2016; Sentir et al., 2020). Thus the current results lend further support to the NVHL rat as a model to better understand SCZ and the increased prevalence of nicotine use.

While additional research is needed to elucidate the exact mechanism, one potential reason for the reduction in context sensitization seen in the NVHL AE group when compared to the NVHL no AE group, may be the impact that alcohol has on developing brain regions. Clinical studies show that alcohol use during adolescence impacts the volume of several brain regions such as the prefrontal cortex (PFC; De Bellis et al., 2005), nucleus accumbens (Thayer et al., 2012), hippocampus (De Bellis et al., 2000; Nagel et al., 2005; Medina et al., 2007), and amygdala (Wilson et al., 2015). Preclinical studies corroborate these findings with alcohol causing numerous anatomical and functional alterations, including decreases in neurogenesis and region-specific brain damage and cell death (Crews et al., 2000; Spear, 2015, 2016). As many of these brain regions play a role in incentive salience, it is possible that disrupted cortical development stemming from alcohol exposure during adolescence dampened the salience of nicotine in the NVHL AE group which prevented the development of context sensitization.

Another neurobiological mechanism that may underlie behavioral changes within the NVHL rat are disruptions in nicotinic acetylcholine receptor (nAChR) function. Extensive literature exists demonstrating that patients with SCZ have disrupted nAChR function and decreased receptor density (Freedman et al., 1995; Breese et al., 2000; Durany et al., 2000; D’Souza and Markou, 2011; Esterlis et al., 2014). These results are supported by a preclinical study showing that NVHL rats have a 12% reduction in nAChR binding in the PFC compared to their sham counterparts (Berg et al., 2015). Furthermore, additional cholinergic alterations exist in this model. In vivo acetylcholine release was hyper reactive to both peripheral and local administration of a dopamine (D)₁ agonist in NVHL rats, and receptor autoradiography showed an increase in muscarinic (M)₁-like receptor binding sites in the PFC (Laplante et al., 2004a). Another study indicated that tail-pinch stress resulted in a significantly greater increase in PFC acetylcholine release in the NVHL rats, which was subsequently blocked by D₁ and D₂ antagonists (Laplante et al., 2004b). Interestingly, nAChRs have been shown to be involved in alcohol-related behaviors where blocking nAChRs partially prevented alcohol-induced locomotor activity (Blomqvist et al., 1992). While some preclinical studies suggest that moderate lengths of alcohol exposure (15–17 days) do not alter nicotinic receptor binding (de Fiebre and Collins, 1993; Ribeiro-Carvalho et al., 2009), chronic alcohol treatment (28 weeks) in rats produced long-lasting reductions in acetylcholine levels, acetylcholinesterase activity, choline uptake, and acetylcholinesterase-positive neurons (Arendt et al., 1988, 1989). Similarly, non-human primates chronically treated with alcohol for 4 weeks had decreased nAChR availability in cortical and thalamic regions (Cosgrove et al., 2010). Though the NVHL AE and Sham AE rats in the current study were only exposed to 14 days of alcohol, there is potential that alcohol exposure during adolescence could alter nAChR function.

Using percentage change as a measure of the amount of nicotine behavioral sensitization, we found that there were no differences between NVHL and sham groups, regardless of whether they received alcohol during adolescence. Our results contrast previously published results showing that NVHL rats (without adolescent alcohol exposure) have enhanced nicotine behavioral sensitization (Berg and Chambers, 2008). A possible explanation for the discrepancies between these studies is the post-weaning housing conditions of the animals. The rats in the current study were singly housed so that alcohol intake during adolescence could be determined for each individual. The rats used in the previously published study (Berg and Chambers, 2008) were pair housed after weaning. Several studies have shown that housing conditions can influence not only the locomotor response to a novel environment, but also the behavioral response to drugs, including nicotine. Rats housed in isolation show increased locomotor response in a novel environment compared to those housed in pairs (Garcia et al., 2017) and those housed in groups (Smith et al., 1997; Cheeta et al., 2001). Furthermore, rats housed in isolation have enhanced sensitization to the locomotor effects of repeated administration of amphetamine (Smith et al., 1997). Additionally, female rats that underwent chronic social stress during adolescence (isolation for 1 h each day and then housed with a new partner) show increased locomotor sensitization in response to amphetamine (Mathews et al., 2008) and nicotine (McCormick et al., 2004). Therefore it is reasonable that isolated housing led to an increase in the nicotine behavioral sensitization of the sham groups, and combined with a potential ceiling effect in the nicotine behavioral sensitization of the NVHL groups, any group differences were masked.

Using measures related to the center zone of the open field arena as a proxy for anxiety-like behaviors (latency to enter the center zone and the duration of time spent in the center zone), we found no differences between NVHL and sham groups, regardless of alcohol exposure (Supplementary Figure 1). The NVHL no AE group did have a significantly increased number of center entrances compared to the other groups, but only during the preinjection phase during the challenge day (Figure 6C). With no other increases in anxiety-like behaviors and the significant increase in both total distance traveled and average velocity, it is likely that the significantly elevated center frequency in the NVHL no AE rats was due to their increased context sensitization. Previous studies assessing anxiety-related behaviors in the NVHL rat have found mixed results based on the method used to measure anxiety. In one study, male NVHL rats demonstrated persistent anxiety as adolescents and adults compared to control rats, spending less time in the central zone of an open field task (Sams-Dodd et al., 1997). However, several other studies found that male and female NVHL rats spend more time in the open arm of an elevated plus maze, suggesting less anxiety (Wood et al., 2003; Beninger et al., 2009). Although the current results found that there were no group differences in anxiety-like behavior using an open field task, future studies assessing anxiety in the NVHL rat should take into account locomotor differences that may confound the results.

In order to assess the effect of nicotine treatment on anxiety-like behaviors and to control for an increase in movement after an injection of nicotine, the ratio of number of center crosses to total distance traveled was calculated for preinjection and postinjection on the challenge day. The significant increase in this ratio during postinjection demonstrates that nicotine had an anxiolytic effect on all groups (Figure 6E). This is consistent with some previous literature showing that 7 days of nicotine treatment (Irvine et al., 2001) or chronic nicotine administered via drinking water (Onaivi et al., 1994) both resulted in anxiolytic effects on elevated plus maze behaviors.

One limitation of the current study was not using both sexes, though there is variability in the literature as to whether a sex difference in nicotine behavioral sensitization exists. Nevertheless, some studies have found that female rats show more locomotor activity in response to nicotine behavioral sensitization (Booze et al., 1999; Harrod et al., 2004), while others find that sex does not have marked influences on this behavior (Kanyt et al., 1999; Ericson et al., 2010). A limited number of studies have used both male and female NVHL rats to assess cognitive abilities (Chambers et al., 1996; Beninger et al., 2009), neurotransmitter release (Beninger et al., 2009), and expression of G-protein coupled receptor kinases (Bychkov et al., 2011). However, to our knowledge, no work has been done exploring sex differences in nicotine behavioral sensitization specifically in the NVHL rat. Another limitation of the current study was the absence of a saline injected group which would serve to control for any handling and injection stress. While this is an important control group, many previous studies have demonstrated that repeated subcutaneous or intraperitoneal injections of saline do not increase locomotor activity (McCormick et al., 2004; Kosowski and Liljequist, 2005; Varvel et al., 2007; Marin et al., 2009; Gomez et al., 2012; Hamilton et al., 2014; Carrara-Nascimento et al., 2020; Trujillo and Heller, 2020). Additional studies corroborate these findings specifically in NVHL rats (Conroy et al., 2007; Berg and Chambers, 2008; Chambers et al., 2013). Therefore, it is highly unlikely that repeated saline injections in our hands would cause behavioral sensitization.

In this study, we found that NVHL rats demonstrated an apparent sensitization to the nicotine paired context, and adolescent alcohol exposure prevented the formation of this context sensitization in the NVHL AE rats. We found that exposure to alcohol during adolescence did not impact the amount of nicotine behavioral sensitization in adulthood. Surprisingly, NVHL rats (regardless of alcohol exposure) did not show increased nicotine behavioral sensitization as had been previously reported, potentially due to post-weaning housing conditions. Nicotine treatment had an anxiolytic effect during the postinjection phase of the challenge day, however, there were no group differences. Future studies could more specifically test the impact of social isolation on nicotine behavioral sensitization and the development of context sensitization in the NVHL rat, as well as expand this work to females.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

The animal study was reviewed and approved by Institutional Animal Care and Use Committee of Dartmouth College.

ES contributed to planning the experimental design and wrote the manuscript. ES, LL, and EB collected the behavioral data. ES and DW performed the NVHL surgeries and DW contributed to editing the manuscript. AH contributed to data analysis and editing the manuscript. EB contributed to the histological assessment. JK and WD contributed to planning the experimental design and editing the manuscript. All authors read and approved the final manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.