Research Findings - Basic Behavioral Research
Progesterone's Attenuation of Cocaine-Primed Reinstatement in Freely Cycling Female Rats is Estrous Cycle Dependent
Cocaine self-administration has been shown to vary with the estrous cycle, and studies in which females are ovariectomized or estrogen is pharmacologically blocked clearly show that ovarian hormones play a role. Estrogen has been shown to have a facilitative effect on cocaine self-administration, including acquisition, escalation (which is commonly used to model the transition from moderate drug use to addiction), and reinstatement (a commonly used model of relapse). In contrast, progesterone has been shown to prevent escalation of cocaine self-administration and to have an inhibitory effect on reinstatement of cocaine-seeking behavior in females. Recently Drs. Matthew Feltenstein and Ron See at the Medical University of South Carolina found an inverse relationship between cocaine-primed reinstatement and plasma progesterone levels in freely cycling female rats across the estrous cycle. In a follow-up study, they directly assessed progesterone's effects on cocaine-primed reinstatement and examined whether its effects varied with the stage of estrous. They found that when administered during diestrus or proestrus, when progesterone is already high, progesterone had no effect. But, when administered during estrus, when progesterone is low, the additional progesterone attenuated cocaine reinstatement. These results complement NIDA-supported laboratory investigations with women showing that cocaine cue-induced craving is inversely related to circulating plasma progesterone levels in women and that experimentally administered progesterone decreases the positive subjective effects cocaine in women but not men. This line of research points to the potential clinical use of progesterone or related compounds in the treatment of cocaine use and addiction. Feltenstein MW, Byrd EA, Henderson AR, See RE. Attenuation of cocaine-seeking by progesterone treatment in female rats. Psychoneuroendocrinology. 2009;34(3):343-52. Epub 2008 Nov 1.
Sex and Age Differences in Sensitivity to Cocaine Conditioned Reward
Cocaine self-administration studies with adult rats have shown that females, compared to males, acquire self-administration faster, show greater escalation of self-administration, greater cocaine-primed reinstatement, and higher motivation for cocaine as measured by the progressive ratio procedure. Recent NIDA-supported research also reveals that female adolescents also acquire cocaine self-administration more rapidly than adolescent males. More recently, Dr. Sari Izenwasser and colleagues at the University of Miami have found that cocaine conditioned reward, as measured by the conditioned place procedure, also is greater in female adolescents than male adolescents, and is greater in adult females than adult males with the order of sensitivity being adolescent females > adult females > adolescent males > adult males. These findings complement other findings showing that the greater sensitivity to cocaine's reinforcing and conditioned reinforcing effects observed in adult females compared to males is not a difference seen only in adulthood, but is present earlier during development, in adolescence. Ongoing research by these investigators is aimed at understand the neurobiological basis of these sex differences. Zakharova E, Wade D, Izenwasser S. Sensitivity to cocaine conditioned reward depends on sex and age. Pharmacol Biochem Behav. 2009;92(1):131-4. Epub 2008 Nov 12.
Gonadal Steroids Mediate Opposite Changes in Cocaine-induced Locomotion across Adolescence in Male and Female Rats
Studies have consistently shown that female rats exhibit greater sensitivity to stimulants than males. This sex difference, however, has not been examined developmentally. Dr. Cynthia Kuhn and her colleagues at Duke University have now characterized the emergence of this difference across development and have examined the role of prepubertal gonadal hormones. In the first study, cocaine was administered at postnatal days 28, 42 and 65. On each of those days, rats received cocaine according to an escalating dose procedure, (designed to simulate binge cocaine effects), and effects on behavioral activation were assessed. In a second experiment with the same protocol intact adults were compared with adults that had been gonadectomized in puberty. Results indicated that across puberty, cocaine responsivity changed in opposite directions in males and females, decreasing in males and increasing in females. Among adults, gonadectomy also had opposite effects in males and females: castration resulted in increased responsivity in males at the highest cocaine dose and ovariectomy decreased responsivity in females at the lower doses. The authors suggest that these data may indicate important organizational effects of testosterone and estrogen during puberty and that these effects may be responsible for sex differences in cocaine responsivity observed in adulthood by virtue of their influence on developing brain dopamine systems. Parylak SL, Caster JM, Walker QD, Kuhn CM. Gonadal steroids mediate the opposite changes in cocaine-induced locomotion across adolescence in male and female rats. Pharmacol Biochem Behav. 2008;89(3):314-23. Epub 2008 Jan 16.
mGlu2/3 Receptors As Targets For Ameliorating Stress-Associated Relapse Risk
Stress is a significant risk factor both in the initiation of drug taking and in relapse to drug use in abstinent drug addicts. Pharmacotherapies with anxiolytic properties therefore offer potential benefit in reducing relapse rates. Dr. Weiss and his colleagues focused their research on group II metabotropic glutamate receptor subtypes (mGluR2/3). They investigated the effects of the selective mGluR2/3 agonist LY354740 (LY), which previous research had demonstrated to have anxiolytic properties, on the shock-probe defensive burying model of anxiety. In this model, the primary index of stress or anxiety is the time spent by rats burying an electrified probe, with increased burying time indicating elevated stress reactivity or anxiety. Weiss and his colleagues tested rats that had been self-administering cocaine under conditions of short or long access to cocaine. The long access condition provides an escalation model of dependence and offers an opportunity to study the behavioral and neurobiological effects of cocaine under conditions that mimic cocaine abuse in humans. The results of three experiments were reported. In Experiment 1, rats were trained to self-administer cocaine, under short (ShA, 1-h) or long (LgA, 6-h) access conditions, or noncaloric food pellets (Ctrl, 1-h), and following 1, 14, 42, or 84 days of abstinence were tested for stress reactivity in the shock-probe defensive burying test. In Experiment 2, experimentally naive rats receiving the mGlu2/3 receptor agonist LY (0, 0.3, 1.0, or 3.0 mg/kg) were tested in the defensive burying test to establish its anxiolytic efficacy in this model. In Experiment 3, rats with a history of ShA vs LgA cocaine self-administration, or a history of operant responding reinforced by noncaloric food pellets, were tested in the defensive burying test, following administration of these same doses of LY at 14 days of abstinence. LgA rats exhibited a two- to threefold increase in defensive burying at 1, 14, and 42 days of abstinence compared to ShA or control animals. LY at 3.0 mg/kg reduced burying in all groups, whereas the 1.0 mg/kg dose reduced burying only in the LgA group. These results reveal that a robust and enduring increase in stress reactivity developed in rats with a history of daily 6-h access to cocaine. They also support the hypothesis that chronic cocaine use produces an increase in stress reactivity that may play a significant role in susceptibility to stress-induced relapse. Finally, these data indicate that mGlu2/3 receptors may be promising treatment targets for stress-induced relapse in cocaine addiction. Aujla H, Martin-Fardon R, Weiss F. Rats with extended access to cocaine exhibit increased stress reactivity and sensitivity to the anxiolytic-like effects of the mGluR 2/3 agonist LY379268 during abstinence. Neuropsychopharmacology. 2008;33:1818-26.
The Dorsal Subiculum and Conditioned Reinstatement of Cocaine-Seeking
Drug associated environmental cues are known to be important elicitors of drug seeking behavior and have a well-documented role in human craving and relapse. Similarly, in animal models, drug-associated cues have been shown to be potent elicitors of drug self-administration in the reinstatement model of relapse. Environment-drug associations typically are established in experiments using multiple drug-cue pairings, but it has now been demonstrated that robust associations, lasting up to one year, can be established after a single 2 hr conditioning session in rats. This persistence of conditioned associations suggests that drug-related learning during an initial cocaine experience may be an important element contributing to continued desire for drug once drug use has begun. Weiss and his colleagues sought to identify brain regions mediating the rapid acquisition of cue conditioning. It is well known that the hippocampus and its major projection, the subiculum play a critical role in the neural substrate for goal-directed behavior. Weiss et al. hypothesized that the subiculum mediates associative learning and plays an important role in behavior controlled by environmental stimuli conditioned with drugs of abuse. The present study investigated whether transient inactivation of the ventral (VSUB) or dorsal (DSUB) subiculum would interfere with the development of cue-induced cocaine-seeking. This hypothesis was tested by reversibly inactivating the VSUB and DSUB with tetrodotoxin (TTX) prior to conditioning rats during a single 2-h period of access to intravenous cocaine. Rats were given 2 h of response-contingent access to intravenous cocaine or saline in the presence of distinct stimuli that served as contextual stimuli associated with the availability and subjective effects of cocaine (S+) versus saline (S- ). Before onset of the sessions, rats received bilateral microinjections of TTX into the VSUB or DSUB. Following extinction, rats were subjected to reinstatement tests in which exposure to the cocaine-, but not saline-associated stimulus produced strong recovery of responding. This effect was completely abolished in rats with transient TTX inactivation of the DSUB during the conditioning session. TTX inactivation of the VSUB during conditioning did not alter the response-reinstating effects of the cocaine cue. The results suggest that functional integrity of the DSUB, but not VSUB, is critical for the acquisition of conditioned cocaine-seeking controlled by contextual stimuli under conditions where such learning occurs during a single conditioning trial. These findings have implications for the transition from initial drug use to addiction and implicate the DSUB as an important neural substrate for the acquisition of drug-related contextual memory, at least during the early stages of this process. Martin-Fardon R, Ciccocioppo R, Aujla H, Weiss F. The dorsal subiculum mediates the acquisition of conditioned reinstatement of cocaine-seeking. Neuropsycho-pharmacology. 2008;33:1827-34.
Novel Approach to Blocking the Toll-like Receptor 4 may be useful in the Treatment of Chronic Pain
Toll-like receptors are an integral part of the immune system and are important in the response to both exogenous and endogenous danger signals. Recently, the toll-like receptor 4 (TLR4) has emerged as a target for the treatment of various diseases, including chronic pain. In a recent study, NIDA-grantee Dr. Linda Watkins and colleagues used a chemical biology approach to disrupt the TLR4-myeloid differentiation factor 2 (MD2) interaction, thereby blocking TLR4 signaling. They demonstrated that short peptides, corresponding to the TLR4-binding loop, can prevent MD2 from binding to TLR4, and that this inhibits the release of proinflammatory cytokines. Proinflammatory cytokine release is responsible for some forms of chronic pain. By specifically blocking the TLR4-MD2 interaction, a cascade of events leading to chronic pain, can be inhibited. Thus, the development of clinically viable small peptides that interfere with the TLR4-MD2 interaction may be clinically useful for treating chronic pain and other disease conditions. Slivka PF, Shridhar M, Lee G, Sammond DW, Hutchinson MR, Martinko AJ, Buchanan MM, Sholar PW, Kearney JJ, Harrison JA, Watkins LR, Yin H. A peptide antagonist of the TLR4-MD2 interaction. ChemBioChem. 2009;10:645-9.
Mechanism for Sex Differences in Pain Perception and Analgesic Responsiveness
Men and women feel pain and respond to analgesics differently. For example, the prototypic analgesic morphine is significantly less effective in women compared with men. In this study, NIDA-grantee Dr. Anne Murphy and colleagues examined some of the potential reasons for this difference in morphine efficacy using a rat model of inflammatory pain. With immunohistochemical techniques, they found that males had a significantly higher expression of mu-opioid receptors (the receptors to which morphine primarily binds) in the ventrolateral periaqueductal gray (PAG) compared with cycling females, and the lowest level of expression was observed in proestrus females. Inflammation of the paw produced thermal hyperalgesia in both males and females that could be significantly reversed in males by a microinjection of morphine into the ventrolateral PAG, whereas this microinjection produced much less analgesia in the proestrus and estrus females. Further, selective lesions of mu-opioid receptor-expressing neurons in the ventrolateral PAG resulted in a significant reduction in the effects of systemic morphine in males only. These data elucidate a mechanism for sex differences in pain perception and analgesic efficacy, and may be of use in the development sex-specific pain treatments. Loyd DR, Wang X, Murphy AZ. Sex differences in mu-opioid receptor expression in the rat midbrain periaqueductal gray are essential for eliciting sex differences in morphine analgesia. J. Neurosci. 2008;28(52):14007-17.
A Sensitizing Regimen of MDMA Causes Enduring Structural Changes in the Accumbens and Prefrontal Cortex
The popular "club" drug MDMA (ecstasy) is a widely used amphetamine derivative, especially by young adults, but relatively little is known about its long-term neurobehavioral effects. A growing body of evidence from animal studies suggests that this drug has neurobiological and behavioral effects that are similar to highly addictive drugs. For example, behavioral studies have shown that repeated MDMA induces behavioral sensitization, conditioned place preference, and drug self-administration. In this study, the investigators gave rats two daily injections of either MDMA or saline vehicle for 3 consecutive days, followed by 4 drug-free days, and repeated this sequence for a total of 3 weeks. Following a 4-week drug-free period, MDMA-pretreated rats displayed behavioral sensitization characterized by a significantly greater locomotor response to a challenge dose of MDMA. At this same time point, rats were sacrificed and their brains processed by a modified Golgi-Cox method for anatomical analysis. The MDMA-pretreated animals showed large increases in spine density and the number of multiple-headed spines on medium spiny neurons in the core and shell subregions of nucleus accumbens. In medial prefrontal cortex, the prelimbic subregion showed increased spine density, while the anterior cingulate subregion showed a change in the distribution of dendritic material instead. These results indicate that long-lasting locomotor sensitization to MDMA is accompanied by reorganization of synaptic connectivity in limbic-cortico-striatal circuitry. The changes in dendritic structure they observed are similar, although not in all brain areas identical to, those found previously in response to cocaine, amphetamine, and nicotine. These structural adaptations, combined with behavioral evidence, suggest that MDMA use may lead to compulsive drug-seeking and drug-taking behavior. Ball KT, Wellman CL, Fortenberry E, Rebec GV. Sensitizing regimens of (+/-)3, 4-methylenedioxymethamphetamine (Ecstasy) elicit enduring and differential structural alterations in the brain motive circuit of the rat. Neuroscience. 2009; Feb 21. [Epub ahead of print]
Cocaine Produces Behavioral Responses in Honey Bees Indicative of Reward
The role of cocaine as an addictive drug of abuse in humans is hard to reconcile with its ecological role as a natural insecticide that prevents herbivory of coca plants. In the past, this paradox has been explained by proposing a fundamental difference in mammalian and invertebrate responses to cocaine, despite the fact that cocaine similarly increases biogenic amine transmission in insects and mammals, and biogenic amines modulate locomotion and reward sensitivity in insects as well as mammals. The question of whether cocaine has reinforcing effects in insects is of importance because insects, such as the fruitfly Drosophila, provide genetic model organisms for the study of other aspects of the neurobiological effects of cocaine. In this study, the investigators show effects of cocaine in honey bees that parallel human responses. Forager honey bees perform symbolic dances to advertise the location and value of floral resources to their nest mates. Thus, the investigators used this "dance language" as a natural bioassay to study the effect of cocaine on reward assessment. When forager bees were treated with a low dose of cocaine, the likelihood and rate of their dancing after foraging increased, although their general locomotor activity did not. This effect is consistent with an interpretation that cocaine caused the bees to overestimate the value of the food resources they had collected. In addition, the experimenters showed that abrupt cessation of chronic cocaine treatment produced a deficit in learning an odor discrimination, similar to learning deficits that occur after cocaine withdrawal in rats. The authors suggest that these similarities likely occur because in both insects and mammals the biogenic amine neuromodulator systems disrupted by cocaine perform similar roles as modulators of reward as well as of motor systems. These analogous responses also propose an alternative solution to the paradox of cocaine reinforcement: ecologically, cocaine is an effective plant defense compound via disruption of herbivore motor control but, because the neurochemical systems targeted by cocaine also modulate reward processing, the reinforcing properties of cocaine may be produced as a "side effect". Barron AB, Maleszka R, Helliwell PG, Robinson GE. Effects of cocaine on honey bee dance behaviour. J Exp Biol. 2009;212(Pt 2):163-8.
Adolescent Anabolic-Androgenic Steroid Exposure Alters Several Neurotransmitters Systems in the Hypothalamus
Richard Melloni and his colleagues have established that chronic anabolic-androgenic steroid (AAS) treatment during adolescence facilitates offensive aggression in male Syrian hamsters. Three recent studies by these investigators explored specific alterations in serotonin, dopamine and glutamate systems that correlate with development of this aggressive phenotype. Serotonin (5-HT) is known to modulate aggressive behavior and has been shown to be altered after AAS treatment. In addition, the 5-HT(2A) receptor has been implicated in the control of aggression. In one of their new studies, they showed that 5-HT(2A) receptor levels and the number of cells expressing this receptor subtype were significantly upregulated in the lateral portion of the anterior hypothalamus (LAH), a brain area thought to be involved in control of aggression. A second study investigated the role of the dopaminergic system in the modulation of AAS-induced aggressive behavior. Aggressive AAS-treated animals showed increased tyrosine hydroxylase immunoreactivity in anterior hypothalamic subnuclei and increased dopamine type 2 (D2) receptor levels in the AH, but decreased D2 levels in the ventrolateral hypothalamus (VLH). These results suggest that alterations in dopamine synthesis and function, together with modifications in D2 receptor expression in the AH, may facilitate AAS-induced aggression. In a third study, they found that glutamatergic cells in the LAH showed a lasting activation following adolescent AAS exposure, as evidenced by increased levels of PAG, the rate limiting enzyme for glutamate synthesis. They also found decreases in afferent innervation from the LAH to the VLH. This decreased connectivity may underlie some of the distinct behavioral peculiarities of AAS-induced aggression found in previous studies. Together, these three studies provide evidence for several neuroplastic mechanisms through which chronic adolescent AAS exposure may facilitate aggressive behavior. They also strengthen the hypothesis that a specific nucleus in the AH, the LAH, is a critical hypothalamic sub-region particularly sensitive to AAS-induced neurodevelopmental effects. Schwartzer JJ, Ricci LA, Melloni RH Jr. Adolescent anabolic-androgenic steroid exposure alters lateral anterior hypothalamic serotonin-2A receptors in aggressive male hamsters. Behav Brain Res. 2009;199(2):257-62. Ricci LA, Schwartzer JJ, Melloni RH Jr. Alterations in the anterior hypothalamic dopamine system in aggressive adolescent AAS-treated hamsters. Horm Behav. 2009;55(2):348-55. Carrillo M, Ricci LA, Melloni RH Jr. Adolescent anabolic androgenic steroids reorganize the glutamatergic neural circuitry in the hypothalamus. Brain Res. 2009;1249:118-27.
Nicotine Withdrawal in Mice Disrupts New, But Not Previously Acquired, Contextual Learning
Research suggests a link between nicotine-induced changes in learning and memory processes and nicotine addiction. Nicotine may facilitate the formation of maladaptive drug-context associations that could lead to drug seeking behavior, and nicotine withdrawal-related disruption of cognitive processes may trigger withdrawal. With this in mind, Drs. Portugal and Gould investigated the effects of nicotine withdrawal on contextual learning that occurred prior to withdrawal and learning that occurred during nicotine withdrawal. To test pre-withdrawal learning, a conditioned place preference procedure (CCP) was used to investigate contextual learning. Mice spent significantly more time in the nicotine-paired environment (e.g., drug context), indicating successful contextual learning. Following CPP testing, mice were surgically implanted with osmotic minipumps that released either nicotine or saline for 12 days. After 12 days, the pumps were removed to initiate spontaneous withdrawal and mice were again tested for CPP to assess whether prior contextual learning was still intact. A nicotine place preference was again observed, revealing that the earlier contextual learning persisted during withdrawal. An hour later, they began training in a fear conditioning paradigm to assess fear-cued conditioning. In this paradigm, freezing in either the training chamber, or a different environment, is used to assess contextual learning. The fear conditioning task paired 30 seconds of a noise cue with mild footshock. Freezing induced by presentation of noise was then used to measure to fear conditioning. Twenty-four hours after training, the mice were tested again, in either the same contextual environment or a different environment and freezing in the absence of noise was recorded. During nicotine withdrawal, only contextual conditioning (assessed on the re-test) was disrupted, with fear conditioning remaining intact. That is, the animals learned the connection between the noise and shock (e.g., fear conditioning), regardless of whether they had been treated with chronic nicotine (and were in withdrawal) or chronic saline (no withdrawal). During the contextual learning assessment (generalized freezing), the animals withdrawn from nicotine displayed less context-dependent freezing. Since the contextual learning that took place prior to nicotine withdrawal remained intact, nicotine withdrawal differentially affected contextual learning depending upon whether acquisition took place before or during withdrawal. This suggests that, during nicotine withdrawal, there are adaptations in neural processes involved in contextual learning. Disruption of learning may play a role in maintaining nicotine addiction, particularly as related to relapse following a period of withdrawal. Portugal GS, Gould TJ. Nicotine withdrawal disrupts new contextual learning. Pharm Biochem Beh. 2009;92:117-23.
Habenula and Interpeduncular α2 and α5 Nicotinic Receptors are Necessary for the Expression of Nicotine Withdrawal
Although it is clear that nicotinic receptors are required for nicotine addiction and subsequent nicotine withdrawal, there are questions as to which nicotinic receptor subtypes, and which brain areas, are involved. Dr. Mariella De Biasi and colleagues investigated the roles of α2 and α5 nicotinic receptors in nicotine withdrawal by comparing mice lacking either α2 or α5 nicotinic receptors with their wildtype littermates. Following chronic nicotine administration via either mini osmotic pump or nicotinated drinking water, systemic administration of mecamylamine (a nicotinic receptor antagonist) was given to precipitate withdrawal. Nicotine withdrawal was calculated using somatic signs, including grooming, scratching, chewing, shaking, cage scratching, head nodding and jumping. While the WT mice displayed significant somatic signs of nicotine withdrawal following mecamylamine administration, neither the α2 nor α5 knockout mice did so. This indicates that α2 and α5 nicotinic receptors are needed for somatic expression of nicotine withdrawal. Investigation into which nicotinic receptor populations in the brain may underlie expression of nicotine withdrawal was then undertaken by administering local injections of mecamylamine following chronic exposure to nicotine. Blocking nicotinic receptors in the habenula or the interpeduncular nucleus precipitated withdrawal as evidenced by somatic withdrawal signs, whereas administration into the cortex, ventral tegmental area or hippocampus did not. This indicates a critical role for nicotinic receptors in the habenulo-interpeduncular system; however conditional KO mice, use of selective ligands, or other targeted technologies are needed to further elucidate the role of particular receptor subtypes. These data suggest that α2 and α5 nicotinic receptors are potential targets for pharmacotherapy for smoking cessation. Salas R, Sturm R, Boulter J, De Biasi M. Nicotinic receptors in the habenulo-interpeduncular system are necessary for nicotine withdrawal in mice. J Neurosci. 2009;29(10):3014-8.