Nicotine addiction is a major preventable risk factor for morbidity and mortality, yet few manage to maintain abstinent when exchanging cigarettes for nicotine replacement therapy (Livingstone-Banks et al., 2019). Defining underlying mechanism associated with nicotine addiction is thus pivotal to outline new strategies for successful nicotine cessation. Experimental studies have shown that drugs of abuse elicit glutamatergic synaptic plasticity that contributes to the reorganization of neural circuits, and putatively, the establishment of addictive behaviors (van Huijstee and Mansvelder, 2014; Christian et al., 2021; Pascoli et al., 2022). Dopamine, which is released in response to nicotine exposure, is a prerequisite for drug-induced plasticity (Belin et al., 2009; Lee et al., 2020; Rivera et al., 2021), and a key regulator of structural and synaptic plasticity (Urakubo et al., 2020; Speranza et al., 2021). In addition, nicotine activates nicotinic acetylcholine receptors (nAChRs), which also has been demonstrated to be important for synaptic plasticity at both inhibitory and excitatory synapses (Dani et al., 2001; Partridge et al., 2002; Adermark, 2011). By activating nAChRs and increasing dopamine levels, nicotine may thus facilitate the induction of synaptic plasticity mechanisms and elicit long-term neuroadaptations, which will outlast the presence of the drug.

Studies performed in male rodents have shown that nicotine especially facilitate synaptic plasticity in the form of endocannabinoid-mediated long-term depression (eCB-LTD; Adermark et al., 2019), and both nicotine self-administration as well as yoked nicotine exposure increase VTA dialysate levels of the cannabinoid 1 receptor (CB1R) agonist 2-arachidonoyl glycerol (2-AG; Buczynski et al., 2013). Antagonists targeting the CB1R furthermore blocks the motivational and dopamine-releasing effects of nicotine (Cohen et al., 2002; Cheer et al., 2007), and mice with a genetic deletion of the CB1R does not show conditioned place preference toward nicotine (Castane et al., 2002). The addictive properties of nicotine may in this extent be connected to facilitation of eCB signaling, which apart from its rewarding properties drive neuronal adaptations that may contribute to the formation of persistent drug-related habits and addictive behaviors (Hilario et al., 2007; Merritt et al., 2008; Hernandez et al., 2014; Bystrowska et al., 2019; Iyer et al., 2022). Repeated exposure to nicotine, however, has been shown to lead to decreased eCB levels in the striatum, without affecting CB1R expression (Gonzalez et al., 2002).

Importantly, smoking cigarettes is in part a gendered activity with sex-specific uptake trends and cessation patterns (Benowitz and Hatsukami, 1998). Smoking is more anxiolytic in women, and women are more likely to smoke in situations associated with stress or in response to negative affect (Perkins et al., 2012; Antin et al., 2017). Interestingly, human studies indicate that there is disparity with regards to nicotine-induced dopamine-release in women and men, where the reward system and ventral striatum is activated by nicotine in men, while dorsal striatal regions are recruited in women (Cosgrove et al., 2014). The dorsal striatum may thus be a key brain region of interest when assessing nicotine-induced neuroplasticity in females. Furthermore, animal studies indicate that female rats display higher tonic 2-AG signaling (Melis et al., 2013), which may affect the responsiveness to the putative nicotine-induced eCB release. Since previous studies outlining nicotine-induced facilitation of eCB signaling has been performed in male rats, the aim of this study was to study acute and sub-chronic effects by nicotine on neurotransmission and synaptic plasticity in the female rat brain. To this end, female Wistar rats were studied using ex vivo electrophysiology and in vivo microdialysis, and nicotine was administered through experimenter-controlled injections. Neurotransmission was monitored in the dorsolateral striatum, a brain region associated with the development of drug-related habits but also linked to neurophysiological responses to nicotine in women (Gerdeman et al., 2003; Belin et al., 2009; Cosgrove et al., 2014).

The data presented here shows that nicotine acutely depresses neurotransmission in a manner that is not driven by CB1R activation or dopamine D2 receptor activation, indicating that dopamine may regulate these acute effects in a partially sex-dependent manner (Licheri et al., 2018). In addition, HFS produced eCB mediated long-standing synaptic plasticity in the DLS of female rats, and nicotine pre-exposure significantly enhanced this form of LTD. This finding is in agreement with previous data retrieved from male rats (Adermark et al., 2019). We also show that repeated exposure to nicotine produces a sustained depression of evoked potentials in the DLS and give rise to a sensitized response to nicotine with regards to both locomotor behavior, as well as dopaminergic and glutamatergic signaling in the DLS. At the same time, our data show that the plasticity facilitating property of nicotine vanishes following repeated nicotine exposure. We also demonstrate that dopamine D2 receptor activation is insufficient to facilitate HFS-LTD in slices from rats previously receiving nicotine. These findings suggest that repeated nicotine exposure produces neuroplastic transformations in the DLS and impaired regulation of stimulation-induced long-term depression, which in the extension may reduce behavioral flexibility and contribute to the development of persistent habits.

Acute administration of nicotine depressed PS amplitude in a manner that was concomitant with a decrease in the probability of transmitter release. This finding is in agreement with previous studies performed on male rats, showing that nicotine acutely depresses the frequency but not amplitude of spontaneous excitatory post synaptic currents in medium spiny neurons in the DLS (Licheri et al., 2018). The exact mechanisms underlying nicotine-induced neuroplasticity in the striatum is not fully understood, but dopamine D2 receptor signaling appears to mediate the down-stream effects of nAChR activation (Licheri et al., 2018; Grieder et al., 2022). Dopamine D2 receptors are also critical for induction of nicotine-induced conditioned place preference (CPP) in mice (Wilar et al., 2019), and the improving effect of nicotine on stress-induced memory impairment (Keshavarzian et al., 2018). Increased levels of dopamine D2 receptor mRNA and receptor binding has also been reported following repeated nicotine administration in experimental studies (Metaxas et al., 2010; Adermark et al., 2016), while a decrease in DAT availability is associated with tobacco addiction in humans (Leroy et al., 2012). Changes in dopaminergic neurotransmission may thus be involved in mediating both acute and protracted neuroplasticity elicited by nicotine. However, while data from male rats has shown that nicotine-induced synaptic depression is occluded by dopamine D2 receptor activation and blocked by dopamine D2 receptor antagonist (Licheri et al., 2018), this was not supported by the data presented here. While the net effect by nicotine on striatal neurotransmission appears to be the same, the mechanisms underlying synaptic depression may thus be partially sex-dependent. Interestingly, while HFS-LTD was enhanced by nicotine, synaptic depression elicited by nicotine alone was potentiated by CB1R antagonist. Since low levels of eCBs has been proposed to act primarily on GABAergic terminals (Uchigashima et al., 2007; Adermark and Lovinger, 2009), it is possible that nicotine stimulates eCB release but that the amount released is insufficient to affect glutamatergic synapses. When combined with HFS, however, eCB-levels are high enough to act on both excitatory and inhibitory terminals (Adermark and Lovinger, 2009). Importantly, the data presented here shows that nicotine-induced synaptic depression engage several signaling pathways and more research is required to fully understand the mechanisms involved.

HFS induced a long-term depression of PS amplitude with a concomitant increase in PPR indicating that synaptic depression is expressed presynaptically. While eCB-signaling has been shown to be important for HFS-LTD in the DLS of male rats (Gerdeman and Lovinger, 2001; Kreitzer and Malenka, 2005; Adermark and Lovinger, 2009), CB1R expression has been shown to be lower in the female mouse striatum compared to males (Liu et al., 2020), and other signaling pathways may thus be recruited. However, HFS-LTD was blocked in slices pre-treated with the CB1R antagonist AM251, showing that eCB-signaling is important for LTD also in the DLS of female rats. Endocannabinoids are produced on demand and the indiction of eCB signaling involves a complex interplay of dopaminergic, glutamatergic and cholinergic receptors (Kreitzer and Malenka, 2005; Wang et al., 2006; Adermark, 2011; Liput et al., 2022). Direct activation of L-type calcium channels produces eCB signaling that is independent on dopamine D2 receptors or mGluR group 1 receptors, suggesting that these signaling pathways are required for facilitating a postsynaptic calcium increase to enable eCB production and release (Adermark and Lovinger, 2007; outlined in schematic drawing, Figure 5E). Quinpirole has previously been shown to robustly increase striatal release of the eCB anandamide in a dopamine D2 dependent manner (Giuffrida et al., 1999). Nicotine could thus facilitate eCB signaling by increasing dopamine D2 receptor signaling, thereby inhibiting cholinergic neurons, and reliving the muscarinic break on post synaptic calcium channels to promote eCB signaling (Wang et al., 2006; Adermark and Lovinger, 2007; Figure 5E). Furthermore, eCB may further suppress activity at striatal synapses by decreasing glutamate transporter activity, resulting in elevated extrasynaptic glutamate concentrations and an indirect suppression of presynaptic glutamate release (Brown et al., 2003; Adermark et al., 2022). While it remains to be determined, the data presented here suggest that nicotine increases eCB signaling, but that HFS is warranted to reach the threshold required for synaptic depression at excitatory terminals (Uchigashima et al., 2007; Adermark and Lovinger, 2009).

Importantly, while drugs of abuse produce synaptic plasticity mechanisms in the acute phase, repeated exposure has been shown to impair eCB signaling (Blanco et al., 2016; Wang et al., 2020). This holds true not only for nicotine (Baca et al., 2013; Xia et al., 2017; Adermark et al., 2019), but also for alcohol and cocaine (Adermark et al., 2011b; Wang et al., 2020). Especially, repeated nicotine exposure appears to impair dopamine D2R-dependent induction of LTD, rather than CB1R signaling per se (Baca et al., 2013; Adermark et al., 2019). In fact, HFS-LTD in male rats have been shown to be restored by quinpirole alone (Adermark et al., 2019), or by quinpirole in combination with the NMDA receptor antagonist APV (Xia et al., 2017). While the data presented here shows that pre-treatment with quinpirole enhance HFS-LTD in slices from vehicle-treated rats, it did not facilitate HFS-LTD in slices from rats receiving sub-chronic nicotine exposure. Interestingly, elevated levels of acetylcholine, which has been reported during nicotine abstinence (Rada et al., 2001), may transform dopamine D2 receptor signaling (Scarduzio et al., 2017). If this is a factor underlying the shifted response to quinpirole remains to be determined.

Repeated exposure to nicotine produced a robust behavioral sensitization, which is in agreement with previous studies (Benwell and Balfour, 1992; Ericson et al., 2010; Hamilton et al., 2014; Morud et al., 2016; Honeycutt et al., 2020). Behavioral sensitization to repeated psychostimulant administration has been proposed to reflect many of the neurochemical changes that are characteristic for drug addiction (Robinson and Berridge, 1993; Steketee and Kalivas, 2011), and is in this regard a well-established model to outline behavioral transformations elicited by drugs of abuse. Behavioral sensitization elicited by psychostimulants has been proposed to be driven by an interplay between dopaminergic and glutamatergic neuroadaptations (Robinson and Becker, 1982; Vezina, 1996; Kim and Vezina, 2002; Bamford et al., 2008; Yoon et al., 2008; Jing et al., 2018; Kim et al., 2022), and neuroplasticity in the dorsal striatum appears to be especially important for establishing these behavioral transformations (Durieux et al., 2012; Kim et al., 2022; Lotfi et al., 2022). The neurophysiological data presented here demonstrated a sensitized response to nicotine with respect to both dopaminergic-and glutamatergic signaling, thereby supporting a role for striatal neuroplasticity in behavioral sensitization. Part of these findings may be linked to increased number of nAChRs, which has been observed in the brains of experimental animals during nicotine treatment and in the brains of human smokers at autopsy (Benwell et al., 1988; Collins et al., 1994; Breese et al., 1997; Alasmari et al., 2021). However, the establishment of behavioral sensitization may also be linked to other forms of striatal neuroplasticity. In fact, inhibition of CB1R has been shown to enhance the motor stimulatory property of the dopamine D2 agonist quinpirole (Giuffrida et al., 1999). This finding suggests that the eCB system may act as an inhibitory feedback mechanism counteracting dopamine-induced facilitation of locomotion. This is especially interesting considering the finding that synaptic depression induced by acute nicotine exposure was potentiated in slices pre-treated with CB1R antagonist. In addition, the data presented here showed reduced HFS-LTD in response to dopamine D2 receptor activation in slices from rats receiving sub-chronic nicotine exposure. It is thus possible that impaired eCB signaling contributes the potentiation of behavioral and neurophysiological response to acute nicotine in rats receiving sub-chronic nicotine exposure.

While nicotine preferentially increases dopamine in the ventral as compared to dorsal striatum in animal studies (Imperato et al., 1986; Shim et al., 2001; Danielsson et al., 2021b), raclopride binding in dorsal striatum of humans have been shown to correlate with subjective measures of the rewarding properties of nicotine (Montgomery et al., 2007). Furthermore, when characterizing the effects of smoking on the mesolimbic dopamine system in humans, dopamine elevations in ventral striatum were consistent with studies performed in men, whereas dopamine responses in women were associated with subregions of dorsal striatum (Cosgrove et al., 2014). Nicotine may thus display sex-specific effects on striatal dopamine transmission. The data presented here show that nicotine elevates the microdialysate concentration of dopamine in the DLS, and that repeated exposure produces a sensitized response to the dopamine-elevating property of nicotine. The sensitized dopamine response may contribute to behavioral transformations and enhanced neuroplasticity in response to nicotine. While sensitized responses to nicotine-induced dopamine elevations have primarily been studied in the nucleus accumbens (Balfour et al., 1998; Vezina et al., 2007), our finding is in agreement with a previous study showing an increased dopamine elevation in dorsal striatum following local administration of nicotine (5 mM) in nicotine exposed rats (Shim et al., 2001). However, more research outlining dopaminergic neurotransmission in dorsal striatal subregions in females is warranted to outline putative sex-specific effects.

Abstinence to nicotine is associated with reduced dopamine levels (Rada et al., 2001; Rahman et al., 2004; Domino and Tsukada, 2009; Rademacher et al., 2016), and withdrawal-induced by the nAChR antagonist mecamylamine has been shown to produce a more pronounced decrease in dopamine levels in the nucleus accumbens of female compared to male rats (Carcoba et al., 2018). However, in vivo microdialysis performed in the DLS revealed no significant decrease in baseline dopamine levels at the time-point assessed here (5 days after the last nicotine injection). This finding may be associated with the fact that drug-induced changes in baseline dopamine levels are more pronounced in the nucleus accumbens compared to the dorsal striatum (Balfour et al., 1998; Danielsson et al., 2021a). In addition, findings may also be highly dependent on the time-point when dopamine levels were assessed (Hirth et al., 2016).

Repeated administration of nicotine resulted in a sustained suppression of evoked potentials in the DLS of female rats. This is partially in agreement with studies performed in male rats, although the neuroadaptations described here arise at an earlier time-point (Adermark et al., 2016). While increased eCB signaling in response to nicotine exposure might contribute to the sustained synaptic depression, decreased PS amplitude in nicotine abstinent rats was not associated with a change in PPR indicating that postsynaptic transformations may underly the sustained synaptic depression. While it remains to be determined what changes that underly the decrease in PS amplitude, impaired glutamate uptake may be one contributing factor. Both nicotine and eCB signaling impair glutamate uptake (Brown et al., 2003; Lim and Kim, 2003a,b; Alasmari et al., 2021), and elevated glutamate levels are reported following repeated nicotine exposure (Carcoba et al., 2018). Reduced glutamate clearance may thus contribute to both acute and sustained neuroplasticity elicited by nicotine (Adermark et al., 2022), possibly through remodeling of synaptic networks and post-translational modifications of glutamate receptors and their interacting proteins (Zheng et al., 2008; outlined in schematic drawing, Figure 5E).

Sex hormones may have the potential to modulate neurohormonal responses to nicotine and influence the psychoactive and reinforcing properties of the drug (Bertrand et al., 1991). Estradiol treatment decreased striatal dopamine D2 receptor binding, and variations in dopamine levels and binding are seen in different phases of the estrous cycle in rats (Bazzett and Becker, 1994; Al Sweidi et al., 2013). Still, there appears to be no estrous cycle dependent changes or sex differences when monitoring nicotine self-administration and reinstatement of nicotine-seeking (Feltenstein et al., 2012; Leyrer-Jackson et al., 2021). In vivo spontaneous activity of dopamine neurons, as well as ex vivo intrinsic and synaptic properties, have also been reported to be similar in male and female rats (Melis et al., 2013), even though there appears to be a sex difference with regards to tonic 2-AG signaling on to dopaminergic neurons (Melis et al., 2013).

There are several limitations with this study. Especially, electrophysiological recordings are conducted ex vivo and the reciprocal connection with other brain regions is thus lost. Field potential recordings also represent the collective activity of hundreds of neurons evoked by electrical stimulation, and differences in direct–indirect pathway neurons can thus not be assessed. Still, eCB-induced LTD is reported in glutamatergic synapses targeting both dopamine D1 and D2 expressing MSNs (Adermark and Lovinger, 2007). Another limitation is that plasticity in the DLS may be especially important during contingent administration and operant behaviors, but nicotine was administered in a non-volitional manner. While the data presented here demonstrates that nicotine produces acute and long-lasting effects on neurotransmission even without the involvement of instrumental learning, it is thus important to further evaluate nicotine-induced neuroplasticity following operant self-administration.

In conclusion, the data presented here demonstrates that nicotine facilitates eCB-mediated LTD and produces striatal synaptic depression by reducing the probability of transmitter release. These findings are similar to what has previously been reported in male rats suggesting that the acute effects mediated by nicotine on excitatory neurotransmission are independent on sex (Licheri et al., 2018; Adermark et al., 2019). However, a sustained depression of evoked PS amplitudes was observed at an earlier time-point in female rats, suggesting that they might be more susceptible to nicotine-induced neuroplasticity (Adermark et al., 2016). In addition, the acute effect of nicotine on PS amplitude was not occluded by dopamine D2 receptor activation, and D2 stimulation did also not facilitate HFS-LTD following sub-chronic nicotine exposure, which also disagrees with findings from male rats (Adermark et al., 2019). Considering the plastic interplay between dopamine and acetylcholine in eCB signaling and glutamate-induced synaptic depression (Calabresi et al., 1999; Wang et al., 2006; Adermark, 2011), these findings might be explained by the proposed sex specificity observed with regards to dopaminergic neurotransmission and dopaminergic regulation of reward-related behavior (Melis et al., 2013; Hynes et al., 2020, 2021). But more studies are required to fully establish sex-specific neuroplasticity associated with repeated nicotine exposure.

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 Göteborgs djurförsöksetiska nämnd, Gothenburg Sweden.

LA designed the study, conducted electrophysiological recordings, assembled figures, and drafted the manuscript. EL handled the animals, conducted behavioral experiments, and assisted during electrophysiological recordings. BS and ME assisted in data interpretation. All authors contributed to the article and approved the submitted version.

This work was supported by the Swedish research council (vetenskapsrådet: Dnr: 2018‐02814, 2020-00559, 2020-01346, 2020-02105), and governmental support under the ALF agreement (ALFGBG-966287).

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.