The Role of Time and Nicotine Dose on Anxiety Measured with Light Enhanced Startle

Two experiments using Light-enhanced startle (LES) examined dose-dependent and timedependent effects of acute nicotine on anxiety. In Experiment 1 rats were exposed to saline, .15 mg/kg, or .40 mg/kg (i.p.) nicotine and 5 minutes later were behaviorally tested in LES. Data suggested that nicotine at both doses was anxiolytic in males but not anxiolytic in females. In females, the higher nicotine dose, .40 mg/kg, was anxiogenic but only during later portions of the test session. In both males and females, within-session variation in LES provided evidence that LES increased in magnitude as time since nicotine administration increased. Therefore, in Experiment 2, longer drug-to-test intervals were applied in order to examine possible timedependent increases in anxiety produced by nicotine. In Experiment 2, rats were exposed to saline or .40 mg/kg nicotine and were behaviorally tested 15 min or 35 min after nicotine administration in LES. Trends in the available data suggested an anxiogenic profile of nicotine when tested 15 min following drug administration but an anxiolytic profile when tested 35 min following drug administration. At the short 5 min drug-to-test interval used in Experiment 1, findings contradict those in other experiments using the social interaction test of anxiety. Collectively, results suggest Light-enhanced startle is sensitive to dose and time-dependent effects of nicotine on anxiety. Possible differences between reflexive and non-reflexive measures of fear and gonadal influences in anxiety expression are discussed. Time and Nicotine in LES 4 The Role of Time and Nicotine Dose on Anxiety Measured in Light Enhanced Startle A recent article published by the American Heart Association indicates that Americans are more likely to use electronic cigarettes than traditional cigarettes because Americans believe that electronic cigarettes are less harmful (American Heart Association, 2019). Though under current debate, even if it is found to be true that e-cigarette vapor contains fewer contaminants than traditional tobacco smoke, the primary neuroactive compound in these devices, nicotine, is still present and can alter typical brain function (American Heart Association, 2019; World Health Organization, 2019). Although nicotine has been studied extensively in the past 40 years the effects of this compound in the brain, which are known to produce anxiety behavior in both humans and animal models, remains unclear (Parrot, 1999). Nicotine is a nicotinic acetylcholine receptor (nAChR) agonist and parasympathetic alkaloid with a high affinity for the α2β4 nAChR subtype (Abou-Donia, 2015). Stimulation of nAChRs by nicotine has been shown to modulate anxiety in stressful situations (Piccioto et al., 2002; Irvine, Cheeta, & File, 2001). Molecularly, nicotine alters the central nervous system by binding to nAChRs which then causes this ligand gated ion channel to turn and open (Wonnacott et al., 2005). The activation of these nAChRs causes an influx of calcium and sodium ions because of said consequential opening of the channel (Kirsch et al., 2016). This molecular cascade can generate or dissipate states of anxiety through its interactions in the basal lateral amygdala (BLA), an area of the brain which is widely recognized for its involvement and production of neural responses to anxiety inducing stimuli (Sharp, 2019). Activation of glutamatergic output neurons of the BLA in part characterize the brain’s response to stressinducing environmental stimuli and play a central role in anxiety. Clinically aberrant activity of the BLA, for example, has been shown to be associated with exaggerated stress responses leading to the progression of anxiety disorders in humans (Graham & Milad, 2011). Importantly, both glutamatergic and GABAergic neurons of the BLA express nAChRs. Stimulation of nAChRs located on glutamatergic neurons of the BLA can directly increase excitatory output of the BLA. Moreover, previous research has shown that increases in glutamatergic responses via nAChR stimulation can contribute to changes in anxiety-like behavior in rats (Ryu et al., 2017). However, nAChRs also expressed on GABAergic afferents to the BLA can increase GABA transmission and modify neural activity by decreasing the glutamatergic excitatory output of the BLA. Through this mechanism, nicotine could potentially decrease anxiety through increased transmission of inhibitory GABA neurons. In summary, both excitatory and inhibitory modulation of the BLA can be mediated by nicotine stimulating nAChRs within the circuitry of the BLA. This is important to nicotine’s relationship with anxiety because the glutamatergic neurons expressing nAChRs and GABAergic neurons expressing nAChRs reveal different routes of activation on BLA that could either increase or decrease anxiety. Animal models are especially valuable in isolating causal influences of nicotine on anxiety. File, Gonzalez, & Andrews (1998) for example, demonstrated in rats that decreases in anxiety measured in the social interaction test, possibly due to nicotine, were blocked by the nonselective nAChR antagonist mecamylamine; revealing a causal role of nAChRs in anxiety. In the social interaction test, anxiety is measured as a reduction in normal prosocial behavior. However, consistent with the dual routes through which nicotine can affect BLA activity described above, there is a current debate in the literature whether nicotine is anxiolytic or anxiogenic in the social interaction test among others. Some studies report nicotine is anxiolytic (Brioni et al., 1993; Costall et al. ,1989; Vale & Green, 1986; Villégier, et al., 2010) whereas others report that nicotine is anxiogenic (Ouagazzal, Kenny, & File, 1999a, 1999b; Morrison, 1969). These paradoxical results could be attributed to the behavioral paradigm used, differences Time and Nicotine in LES 5 in dose and route of administration of nicotine, and the nicotine-to-test interval used, among other experimental parameters (for a review see Picciotto, Brunzell, & Caldarone, 2002; see Morissette et al., 2007 for a related discussion of interrelationships between nicotine and anxiety in humans). Assessment of the delay between nicotine administration and behavioral (or neural) testing is a critical factor because nicotine can modulate the release of neurotransmitters in different parts of the brain and it is likely that nAChRs have different time courses of inactivation and activation which could have time-dependent effects on brain systems important to anxiety (Picciotto et al., 2002). Interestingly, some studies using the same behavioral task report differing effects of nicotine when the nicotine-to-test interval is varied. In one striking example, Irvine, Cheeta, and File (1999) varied the nicotine-to-test delay and reported in the social interaction test with rats that the same dose of nicotine (.1 mg/kg) had anxiogenic effects with a 5 min nicotine-to-test interval, but anxiolytic effects at a longer 30 min nicotine-to-test interval. Thus, the same dose of nicotine had opposite effects on behavior. The current research systematically examines the impact of a nicotine-to-test interval similar to that of Irvine et al. (1999) but examines anxiety in light-enhanced startle, a validated animal model of anxiety (Walker & Davis, 1997). Unlike Irvine et al. (1999) the current research additionally examines the effect of nicotine dose on anxiety, and explores possible sex differences. In the light-enhanced startle (LES) paradigm the rat’s acoustic startle reflex to brief but loud bursts of white noise is measured in a dark setting and then is measured again in the presence of a bright light, an innately aversive stimulus to rodents (Walker & Davis, 1997). In this model, high illumination levels are thought to increase anxiety and thereby increase the magnitude of the acoustic startle reflex in light compared to dark sessions (Walker & Davis 1997). This paradigm advantageously provides a way to assess acoustic startle response to unconditioned fear stimuli implicated in anxiety (de Jongh et al, 2003). Additionally, research has shown that increases of startle reflex in LES reflect an influence of higher anxiety levels while low levels of startle reflect states of lower anxiety (Walker & Davis 2001). LES also provides further advantages over a related paradigm, fear-potentiated startle (FPS), another model of anxiety, because LES reflects unlearned fear to unconditioned stimuli whereas FPS is specifically designed to assess anxiety responses to learned, conditioned fear stimuli. As a result, LES can be repeatedly tested without contaminating factors of learned fear extinction or conditioned sensitization (Walker & Davis 1997; 2001). Finally, LES serves a valuable tool because it measures an unbiased and ‘pure’ form of anxiety. As described, LES is not subject to extinction in repeated testing thereby allowing the behavioral measure of anxiety in LES to be uncoupled from learning related changes in anxiety behavior. Additionally, LES, as a startle paradigm using the acoustic startle reflex as primary response measure, engages relatively simple hindbrain circuits (Walker & Davis 1997; 2001) unlike anxiety measured in the social interaction test of Irvine et al. (1999) which is known to recruit higher brain processing centers including the hippocampus (File, Kenny, & Ouagazzal, 1998) and frontal cortex (Ko, 2017). Tests of anxiety in the social interaction test involve choice and decision making whereas anxiety measured in LES involves innate unlearned reflexes. Examining nicotine’s effect on anxiety specifically in LES may therefore be of further interest because it will permit comparison of nicotine’s impact on reflexive and non-reflexive aspects of fear. The present research had two goals. Experiment 1 examined the effect of nicotine dose on the magnitude of LES in order to clarify anxiolytic vs. anxiogenic profile of nicotine on an