Adolescent male rats are less sensitive than adults to the anxiogenic and serotonin-releasing effects of fenfluramine
Highlights
► Fenfluramine was more anxiogenic to adult male rats than adolescents. ► Adolescents had lower fenfluramine-stimulated cortical serotonin release. ► Lower tissue serotonin stores may limit the effects of fenfluramine in adolescents. ► Serotonin-releasing drugs of abuse could also be less anxiogenic to adolescents.
Introduction
Adolescence is the period of transition from childhood to adulthood (Spear, 2000). This transitional period includes the time from around 12 to 18 years of age in humans (reviewed in Spear, 2000). In rodents, adolescence encompasses postnatal days 28–42 (PN28–42), though adulthood is not considered to begin until around PN60 (reviewed in McCutcheon and Marinelli, 2009; Spear, 2000). Behavior changes during adolescence, with risk taking, novelty seeking, and social behavior expressed at higher levels than in childhood or adulthood (Stansfield and Kirstein, 2006; Steinberg et al., 2008, 2009; reviewed in Spear, 2000). Impulsive, risk taking behavior is part of normal development, but also contributes to major causes of adolescent injury and mortality such as reckless driving, suicide, unsafe sexual behavior, and experimentation with drugs (Chen and Kandel, 1995; Eaton et al., 2010; SAMHSA, 2011; Steinberg, 2008; reviewed in Spear, 2000).
Immature function of the neural circuits that mediate goal directed behavior contributes to adolescent risk taking. The balance between systems mediating approach to rewarding stimuli and avoidance of aversive stimuli may be biased toward approach during adolescence (reviewed in Ernst and Fudge, 2009; Ernst et al., 2006). Prefrontal cortical regulation of limbic brain regions is immature, limiting the regulation of these approach and avoidance drives (reviewed in Casey et al., 2011; Ernst and Fudge, 2009; Ernst et al., 2006; Steinberg, 2010; Sturman and Moghaddam, 2011). Immaturity of dopaminergic and serotonergic function in the forebrain may also contribute to this approach/avoidance imbalance in adolescents (reviewed in Chambers et al., 2003; Crews et al., 2007; Ernst et al., 2006). Serotonin is an important mediator of behavioral inhibition in response to aversive situations, and low central serotonergic function has been associated with risk taking, impulsivity, and aggression (Brown et al., 1979; Crockett et al., 2009; Higley and Linnoila, 1997; Higley et al., 1996; Mehlman et al., 1994; Soubrie, 1986; Virkkunen et al., 1995). Serotonin also contributes to the aversive effects of some drugs of abuse, and adolescents are less sensitive to aversive effects of drugs in animal models (Ettenberg and Bernardi, 2006, 2007; Ettenberg et al., 2011; Infurna and Spear, 1979; Jones et al., 2009, 2010; Rocha et al., 2002; Schramm-Sapyta et al., 2006; Serafine and Riley, 2010). Lower serotonergic function in adolescents could therefore contribute to increased risk taking behavior and reduce the aversive effects of drugs of abuse. These effects could factor into the increased experimentation with drugs seen during adolescence (Chen and Kandel, 1995; SAMHSA, 2011).
Animal studies suggest that forebrain serotonergic function during early adolescence may be lower than in adults, especially in the cortex. While serotonin receptor expression, dorsal raphe firing rates, and the anatomic pattern of serotonergic innervation appear to be mature by adolescence, neurochemical markers of presynaptic serotonergic function increase between adolescence and adulthood (Beique et al., 2004; Daval et al., 1987; Garcia-Alcocer et al., 2006; Lanfumey and Jacobs, 1982; Lidov and Molliver, 1982; Miquel et al., 1994; Pranzatelli and Galvan, 1994; Vizuete et al., 1997; Waeber et al., 1996, 1994). Serotonin transporter (SERT) binding is lower in the cortex of early adolescent rats (PN28–35), and some studies show lower SERT binding in subcortical regions such as the amygdala and striatum (Dao et al., 2011; Galineau et al., 2004; Moll et al., 2000; Tarazi et al., 1998). Serotonin tissue content and synaptosomal uptake are also lower in the cortex and striatum of early adolescent rats compared to adults (Kirksey and Slotkin, 1979; Loizou, 1972; Loizou and Salt, 1970; Mercugliano et al., 1996). Studies during later adolescence have reported more adult-like serotonin function. Older adolescent rats (PN45–50) and adults have similar baseline and methamphetamine-stimulated extracellular serotonin in medial prefrontal cortex (mPFC) (Staiti et al., 2011). SERT binding and serotonin uptake are also mature by this age (Kirksey and Slotkin, 1979; Tarazi et al., 1998). These studies show that neurochemical markers of presynaptic serotonergic function reach adult levels during the adolescent period, and that these markers are lower than in adults during early adolescence from PN28 to PN35.
While the ontogeny of forebrain serotonergic innervation has been described in young adolescents, serotonin release in response to pharmacologic or behavioral stimuli has not been evaluated over this critical developmental window. Additionally, the ability of serotonergic drugs to influence some behaviors has been studied in adolescents of several species, but a direct comparison of serotonergic regulation of behavioral inhibition in adolescents and adults has not been reported (Higley et al., 1996; LeMarquand et al., 1998; Mehlman et al., 1994; Zepf et al., 2008). The purpose of this study is to address this gap in our understanding of how serotonin regulates behavior during adolescence by assessing behavioral and neurochemical responses to pharmacologic challenge of the serotonergic system in adults (PN67–74) and early adolescents (PN28–34). We used the serotonin-releasing drug fenfluramine to compare the ability to mobilize serotonin stores in adults and early adolescents. Fenfluramine mobilizes serotonin stores by a similar mechanism as drugs of abuse such as methamphetamine and methylenedioxymethamphetamine (MDMA), but is more selective for the serotonergic system than these drugs (Rothman et al., 2001; reviewed in Sulzer et al., 2005). The behavioral effects of fenfluramine treatment were evaluated in the light/dark (LD) and the elevated plus maze (EPM) tests for anxiety-like behavior. Unconditioned tests for anxiety-like behavior such as the LD and EPM are thought be an ethologically relevant model of risk taking behavior because they measure inhibition of species-typical behaviors in novel, aversive, and potentially risky environments (Harro, 2002; Macri et al., 2002; Olausson et al., 1999). We then used microdialysis to assess the effect of fenfluramine and potassium on extracellular serotonin in the mPFC, and further investigated presynaptic serotonin function by measuring serotonin content, synthesis, innervation density, and SERT levels in the prefrontal cortex, amygdala, and hippocampus.
Section snippets
Animals
Young adult (PN60–63) and juvenile (PN21) male Sprague–Dawley (CD) rats were purchased from Charles River Laboratories (Raleigh, NC). The rats were housed in ventilated plastic cages (Techniplast USA, Exton, PA) or standard rat cages (Allentown Caging, Allentown, NJ) with corn cob bedding on a 12:12 h light/dark cycle with lights on at 06:00 and lights off at 18:00. All rats were allowed to acclimate to our AALAC accredited facility for at least 7 days before behavior testing or collection of
Light/dark test
The time spent in the light compartment and the latency to emerge into the light were used as measures of anxiety-like behavior, and the total distance traveled was used to assess locomotion (Morley et al., 2005; Schramm-Sapyta et al., 2007). Fenfluramine increased anxiety-like behavior in both ages (n = 12 per experimental group) as shown by a reduction in time spent in the light compartment (Fig. 2A) [main effect of Treatment, F(1,39) = 77.92, p < 0.001]. This effect was significantly greater
Discussion
This study shows that adolescent male rats are less sensitive than adult males to the anxiogenic effects of the serotonin-releasing drug fenfluramine, and produces neurochemical data which suggest this behavioral effect may result from lower fenfluramine-stimulated serotonin release in adolescents. Fenfluramine did not induce anxiety-like behavior as effectively in adolescent rats as in adults in either the LD test or EPM. The anxiogenic effects of fenfluramine in adult rats are consistent with
Acknowledgment
This work was supported by National Institute on Drug Abuse grants DA019114 and 1F31DA032532. The authors wish to thank Sam Johnson and the Duke Light Microscopy Core Facility for technical help with imaging and Jacob Jacobsen for technical help with microdialysis.
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