Limitations of using adolescent rodent models for complex human disorders

We have only tested a couple possible factors known to be involved in addiction, but cer- tainly other factors are involved. Moreover, adolescent human drug vulnerability may end up not being biologically based. Other factors known to be important for human drug abuse are social factors such as peer-pressure, cognitive factors such inhibition and impulsivity, environmental factors such as access to drugs, and internal factors such as response to stress. Also, we have initially focused on only one way of reinstating drug- seeking behavior, context or discrete cues, but stress- and the drug itself are also used to induce reinstatement of drug-seeking behavior. And maybe continued drug intake is im- portant, as our adolescent rats only take drugs during the short period of adolescence, and not continued into adulthood.

Some characteristics of rodent adolescence may limit its use as a model for hu- man adolescence. Adolescence in rodents is quiet short, spanning only approximately 20 days (Spear 2000 for review). Thus, experiments intended to take place during the ado- lescent period in rodents must be modified to fit in this short time span and may therefore not be as thorough, possibly leading to inconclusive interpretations. For example, full dose effect functions during self-administration are needed for accurate conclusions re- garding the reinforcing potency and efficacy of the test drug, but rats usually take approx- imately five days to learn to acquire the association of the lever and the infusion of the

drug, and acquisition must come before any test of dose effects. It is possible to pre-train rats to lever press for food pellets to speed up acquisition, but then food deprivation is used to motivate the rats, which adds possible confounding motivational states. It is also possible to speed up testing phases by running two self-administration sessions per rat per day, but that would also reduce the number of rats tested at one time and drug effects from the first test of the day might carry over to the second test of the day. Another tech- nical drawback is the need to modify the i.v. catheters for the initial small size of adoles- cent rats, but also allow for the rapid growth spurt. Our lab can only reliably catheterize adolescent rats starting as early as P28, which means worthwhile young adolescent days are spent recovering from surgery. Also, great care must be taken to maintain i.v. cathe- ters for long periods, yet despite caution most labs using i.v. catheters consistently lose approximately 10% of rats to catheter malfunction.

6.7 Future Directions

Guided by the framework of current literature and the present results, a number of future directions are worth pursuing based on factors known to be important for reinforcement and reinstatement of drug-seeking in adult subjects and development of the nervous sys- tem in adolescents. First, there seems to be a dichotomy in adolescent vs. adult reins- tatement of cocaine-seeking (Anker and Carroll 2010), in that adolescent- vs. adult-onset male rats exhibit less context/cue-induced, but more cocaine- and yohimbine-induced reinstatement of drug-seeking. It would be worthwhile to test if this same pattern of reinstatement behavior was exhibited in adolescent- vs. adult-onset rats with a history of heroin or morphine self-administration. Adolescents may be exceptionally vulnerable to stress (Spear 2009), and in adult rats modulation of the central nervous stress system im-

pacts heroin-seeking behavior (Banna et al. 2010; Shaham et al. 2000; Zhou and Kalivas 2008). Also of interest, in adult male rats the context in which heroin vs. cocaine or am- phetamine are self-administered (home cage vs. separate chamber and room) seems to important for amount of drug intake and Fos mRNA levels in the brain (Celentano et al. 2009), but it is not known if this environmental factor also influences age-dependent drug-seeking behavior. As mentioned above, maybe drug use beginning during adoles- cence and continuing into adulthood would be a more influential drug history pattern for adolescent vulnerability and would be a better model of adolescent-onset human drug ad- dicts, compared to our current short two week self-administration conditions. Indeed, long-term self-administration (3 months) seems be important for the appearance of hu- man addiction-like behaviors in rats (Deroche-Gamonet et al. 2004).

Our lab has yet to test which neurotransmitter systems are regulating age-

dependent drug-seeking (i.e. opioidergic, dopaminergic and glutamatergic systems within reward and reinforcement areas in the brain). Obvious candidates exist known to control drug-seeking in adult rodents that also go through dramatic changes during the adolescent developmental period. In regards to the opioid system [maintenance pharmacotherapy with opioid ligands methadone, naltrexone, or buprenorphine are currently the gold stan- dard treatment for human opiate addicts], mu opioid receptor efficiency is blunted during early adolescence compared to adulthood in rats (Talbot et al. 2005), and in adult rats me- thadone maintenance reduces heroin- and cocaine-induced reinstatement of drug-seeking (Leri et al. 2004). In regards to the dopamine system, dopamine D1 receptors are over- expressed and associated second messenger system activity is blunted during adolescence (Andersen 2002; Andersen and Teicher 2000; Brenhouse et al. 2008), and in adult rats

injection of the dopamine D1-family receptor antagonist (SCH 23390) into the dorsola- teral, but not dorsomedial, striatum attenuates heroin-seeking behavior (Bossert et al. 2009).

In regards to the glutamate system, adolescence and puberty are intimately linked to proper glutamate system function (Parent et al. 2005). Electrophysiological data sug- gest greater AMPA receptor contribution to excitatory postsynaptic currents (EPSCs) in the NAcc among adolescents vs. adults (Kasanetz and Manzoni 2009), but in PFC in spe- cific populations of interneurons, NMDA- but not AMPA-mediated current might contri- bute more in adolescents vs. adults (Wang and Gao 2009). In the PFC, as well, increased basal expression of specific isoforms of AMPA receptor subunits GluR1/3 in adolescent vs. adult rats was observed (Stine et al. 2001), whereas another study suggested possible more GluR1 but less GluR2-4 subunit expression in younger rats (although this later comparison was made in younger P21, vs. P110 adults; Talos et al. 2006). Also, AMPA receptors exhibit age-, dose-, and brain area-dependent changes following adolescent vs. adult administration of nicotine (Adriani et al. 2004), suggesting that age-at-onset of drug intake is important for some drug-related long-term glutamate system plasticity. In adult rats systemic and intra-VTA injections of the glutamate group II metabotropic receptor agonist (LY379268) reduces glutamate release and attenuates heroin-seeking in adult rats (Bossert et al. 2004). Also, glutamate AMPA receptor subunit ratio/activity within mPFC and NAcc are critical for drug-seeking behavior in adult rats; GluR2 and GluR3 subunit downregulation were associated with cue-induced heroin-seeking and blocking GluR2 endocytosis in infralimbic, but not prelimbic, mPFC attenuates cue-induced he- roin-seeking (Van den Oever et al. 2008); and blockade of GluR2-lacking receptors in

NAcc attenuates “incubation” of cocaine-seeking (Conrad et al. 2008). Furthermore in adult rats, treatment with N-Acetylcystine, which restores cystine–glutamate tone after repeated drug use, reduces extinction responding and cue- and heroin-induced (Zhou and Kalivas 2008) and cocaine-seeking behavior (Moussawi et al. 2011).

Lastly, the cellular and molecular mechanisms of neuroplasticity have emerged as targets for drug addiction research, but little is known about neuroplasticity in rats that self-administer drugs as adolescents. Our lab has begun to explore expression of plastici- ty genes, such as BDNF, Arc and Erk, during abstinence and reinstatement of drug- seeking in rats that self-administered cocaine as adolescents (Li and Frantz in prepara- tion). It would be interesting to find out if the effects of cocaine self-administration in adolescent rats on neuroplasticity genes can generalize to rats with a history of adolescent morphine or heroin self-administration, especially during protracted withdrawal.