DAT isn't all that: cocaine reward and reinforcement require Toll-like receptor 4 signaling.

The initial reinforcing properties of drugs of abuse, such as cocaine, are largely attributed to their ability to activate the mesolimbic dopamine system. Resulting increases in extracellular dopamine in the nucleus accumbens (NAc) are traditionally thought to result from cocaine's ability to block dopamine transporters (DATs). Here we demonstrate that cocaine also interacts with the immunosurveillance receptor complex, Toll-like receptor 4 (TLR4), on microglial cells to initiate central innate immune signaling. Disruption of cocaine signaling at TLR4 suppresses cocaine-induced extracellular dopamine in the NAc, as well as cocaine conditioned place preference and cocaine self-administration. These results provide a novel understanding of the neurobiological mechanisms underlying cocaine reward/reinforcement that includes a critical role for central immune signaling, and offer a new target for medication development for cocaine abuse treatment.

Opioid activation of toll-like receptor 4 contributes to drug reinforcement.

Opioid action was thought to exert reinforcing effects solely via the initial agonism of opioid receptors. Here, we present evidence for an additional novel contributor to opioid reward: the innate immune pattern-recognition receptor, toll-like receptor 4 (TLR4), and its MyD88-dependent signaling. Blockade of TLR4/MD2 by administration of the nonopioid, unnatural isomer of naloxone, (+)-naloxone (rats), or two independent genetic knock-outs of MyD88-TLR4-dependent signaling (mice), suppressed opioid-induced conditioned place preference. (+)-Naloxone also reduced opioid (remifentanil) self-administration (rats), another commonly used behavioral measure of drug reward. Moreover, pharmacological blockade of morphine-TLR4/MD2 activity potently reduced morphine-induced elevations of extracellular dopamine in rat nucleus accumbens, a region critical for opioid reinforcement. Importantly, opioid-TLR4 actions are not a unidirectional influence on opioid pharmacodynamics, since TLR4(-/-) mice had reduced oxycodone-induced p38 and JNK phosphorylation, while displaying potentiated analgesia. Similar to our recent reports of morphine-TLR4/MD2 binding, here we provide a combination of in silico and biophysical data to support (+)-naloxone and remifentanil binding to TLR4/MD2. Collectively, these data indicate that the actions of opioids at classical opioid receptors, together with their newly identified TLR4/MD2 actions, affect the mesolimbic dopamine system that amplifies opioid-induced elevations in extracellular dopamine levels, therefore possibly explaining altered opioid reward behaviors. Thus, the discovery of TLR4/MD2 recognition of opioids as foreign xenobiotic substances adds to the existing hypothesized neuronal reinforcement mechanisms, identifies a new drug target in TLR4/MD2 for the treatment of addictions, and provides further evidence supporting a role for central proinflammatory immune signaling in drug reward.

Effects of chronic caffeine exposure during adolescence and subsequent acute caffeine challenge during adulthood on rat brain serotonergic systems.

Caffeine is the most commonly used drug in the world. However, animal studies suggest that chronic consumption of caffeine during adolescence can result in enhanced anxiety-like behavioral responses during adulthood. One mechanism through which chronic caffeine administration may influence subsequent anxiety-like responses is through actions on brainstem serotonergic systems. In order to explore potential effects of chronic caffeine consumption on brainstem serotonergic systems, we evaluated the effects of a 28-day exposure to chronic caffeine (0.3 g/L; postnatal day 28-56) or vehicle administration in the drinking water, followed by 24 h caffeine withdrawal, and subsequent challenge with caffeine (30 mg/kg; s.c.) or vehicle in adolescent male rats. In Experiment 1, acute caffeine challenge induced a widespread activation of serotonergic neurons throughout the dorsal raphe nucleus (DR); this effect was attenuated in rats that had been exposed to chronic caffeine consumption. In Experiment 2, acute caffeine administration profoundly decreased tph2 and slc22a3 mRNA expression throughout the DR, with no effects on htr1a or slc6a4 mRNA expression. Chronic caffeine exposure for four weeks during adolescence was sufficient to decrease tph2 mRNA expression in the DR measured 28 h after caffeine withdrawal. Chronic caffeine administration during adolescence did not impact the ability of acute caffeine to decrease tph2 or slc22a3 mRNA expression. Together, these data suggest that both chronic caffeine administration during adolescence and acute caffeine challenge during adulthood are important determinants of serotonergic function and serotonergic gene expression, effects that may contribute to chronic effects of caffeine on anxiety-like responses.

Interactive effects of nicotine and alcohol co-administration on expression of inducible transcription factors in mouse brain.

Nicotine and alcohol are abused substances that are often used concurrently. Despite their combined usage, little is known about how they interact to produce changes in behavior and neural activity. Two experiments were conducted to identify interactions on both behavior and neural targets resulting from the co-administration of nicotine and alcohol. In Experiment 1, male C57BL/6J mice were administered saline, alcohol (2.4 g/kg, i.p.), nicotine (0.5 mg/kg, i.p.) or an alcohol/nicotine mixture and returned to their home cage. In Experiment 2, a higher dose of nicotine (1.0 mg/kg, i.p.) was included and animals were exposed to a novel environment. Several behavioral measures were analysed during novelty exposure. Immunohistochemical detection of inducible transcription factors (c-Fos and Egr1) was used in both experiments to identify changes in neural activation. Behavioral results suggested that the drugs were interacting in the production of behaviors. In particular, alcohol produced locomotor stimulation while it suppressed counts of rearing and leaning. When co-administered, nicotine appeared to counteract the alcohol-enhanced locomotor activity. Several brain regions were observed to have altered transcription factor expression in response to the different drug treatments, including amygdalar, hippocampal and cortical subregions. In a subset of these brain areas, nicotine and alcohol counteracted one another in the expression of transcription factors. These results identify several interactive target sites within the hippocampus, extended amygdala and cortical regions. The interactions appear to be a result of antagonizing actions of nicotine and alcohol. Finally, the results suggest that the combined use of nicotine and alcohol may offset the effects of the drug administered independently.

Strain differences in urocortin expression in the Edinger-Westphal nucleus and its relation to alcohol-induced hypothermia.

The Edinger-Westphal nucleus is the primary source of urocortin in rodent brain. Mapping of inducible transcription factors has shown that the Edinger-Westphal nucleus is preferentially sensitive to ethanol self-administration. In the present study we have immunohistochemically compared expression of urocortin and c-Fos in naive and ethanol-treated C57BL/6J and DBA/2J mouse inbred strains. We found that C57BL/6J mice possess significantly higher numbers of urocortin-expressing cells in the Edinger-Westphal compared to DBA/2J mice. Subsequent histological analysis confirmed a lower number of large neurons in the DBA/2J Edinger-Westphal nucleus. Surprisingly, despite the differences in structure, no strain differences were observed in the number of c-Fos-containing cells after acute (0.6-4.8 g/kg, i.p.) and repeated (2.4 g/kg, 14 days, one injection/day) administration of ethanol. Double-label immunohistochemistry showed that ethanol-induced c-Fos expression is present in different sets of Edinger-Westphal cells between the strains. Specifically, expression of c-Fos in C57BL/6J mice is preferentially induced in urocortin cells, while c-Fos in DBA/2J mice occurs in a mixed population of cells. Behavioral analysis of the B6D2 F2 intercross, a heterogeneous mouse strain, showed that the number of urocortin cells is positively correlated with basal temperatures and ethanol-induced hypothermia. Involvement of the Edinger-Westphal in alcohol-induced hypothermia is further confirmed by analysis of urocortin cells in the HOT/COLD selected lines. These results provide evidence that C57BL/6J and DBA/2J mice have structural differences in the Edinger-Westphal that can result in activation of different populations of neurons upon alcohol intoxication contributing to differential thermoregulation between these inbred strains.

ITF expression in mouse brain during acquisition of alcohol self-administration.

Expression of inducible transcription factors (ITFs) c-Fos and FosB was investigated during acquisition of alcohol drinking in C57BL/6J mice. A slight but statistically significant increase in c-Fos expression was found in the Edinger-Westphal nucleus (EW) of animals consuming 2% ethanol/10% sucrose for the first time. Stronger expression of c-Fos in EW was found in animals repeatedly consuming ethanol-containing solutions. These findings underscore the potential importance of EW in alcohol-related behaviors.

Alcohol drinking produces brain region-selective changes in expression of inducible transcription factors.

Mapping the effects of alcohol consumption on neural activity could provide valuable information on mechanisms of alcohol's effects on behavior. The present study sought to identify effects of alcohol consumption on expression of inducible transcription factors (ITFs) in mouse brain. C57BL/6J mice were trained to consume 10% ethanol/10% sucrose solution during a 30-min limited access period. Control animals were given access to 10% sucrose solution or water. Following the final day of the procedure, animals were sacrificed and immunohistochemical analyses were performed for three ITFs (c-Fos, FosB, and Zif268). Alcohol-consuming animals had increased ITF expression in several brain areas. Specifically, c-Fos was significantly induced in the nucleus accumbens core (AcbC), the medial posteroventral portion of the central nucleus of the amygdala (CeMPV), and the Edinger-Westphal nucleus (EW). Expression of c-Fos was significantly lower in the dentate gyrus of alcohol-consuming animals vs. sucrose-consuming animals. However, it was not significantly different from the water controls. Induction of c-Fos in AcbC, CeMPV and EW was significantly related to blood alcohol concentrations (BAC). Furthermore, FosB expression in the CeMPV and the EW was also significantly higher in the alcohol-consuming animals vs. water controls. FosB expression in the EW was significantly related to BAC. The significance of these results is two-fold. First, our experiments demonstrate that ITF mapping is an effective strategy in identifying alcohol-induced changes following voluntary consumption. Second, they suggest a relationship between ITF expression in AcbC, CeMPV and EW and the level of alcohol intoxication.

Cocaine- and alcohol-mediated expression of inducible transcription factors is blocked by pentobarbital anesthesia.

Identifying the neurocircuitry involved in behavioral responses to drugs of abuse is an important step towards understanding the mechanisms of drug addiction. The present study sought to distinguish brain regions involved in pharmacological effects of cocaine and ethanol from secondary effects by administering these drugs in the presence or absence of pentobarbital anesthesia. Changes in neuronal activity were assessed by immunohistochemical analysis of expression of an inducible transcription factor (ITF), c-Fos, in the brain of rats habituated to repeated pentobarbital anesthesia or saline administration. Cocaine administration (15 mg/kg, i.v.) in non-anesthetized animals produced a strong induction of c-Fos in the striatum and large number of other brain areas. Ethanol administration (2 g/kg, i.p.) induced c-Fos in a smaller number of characteristic brain areas, including the central nucleus of amygdala and paraventricular nucleus of hypothalamus. However, neither of these drugs was able to induce c-Fos in pentobarbital-anesthetized rats (50 mg/kg, i.v.). The suppressive effects of pentobarbital were not specific to c-Fos, such that pentobarbital also suppressed expression of ITFs FosB and Egr1 in the striatum of cocaine-treated rats. On the other hand, pentobarbital by itself strongly induced c-Fos expression in the lateral habenula of saline-, cocaine-, and ethanol-injected rats. It is not clear whether the suppressive effects of anesthesia on ITF expression in other areas are mediated by activation of lateral habenula, or are independent of this event. Our data suggest that in the absence of conscious awareness of drug-associated cues, cocaine and alcohol activate only a fraction of the neural elements engaged in the unanesthetized state.

Distinct patterns of DeltaFosB induction in brain by drugs of abuse.

The transcription factor DeltaFosB accumulates and persists in brain in response to chronic stimulation. This accumulation after chronic exposure to drugs of abuse has been demonstrated previously by Western blot most dramatically in striatal regions, including dorsal striatum (caudate/putamen) and nucleus accumbens. In the present study, we used immunohistochemistry to define with greater anatomical precision the induction of DeltaFosB throughout the rodent brain after chronic drug treatment. We also extended previous research involving cocaine, morphine, and nicotine to two additional drugs of abuse, ethanol and Delta(9)-tetrahydrocannabinol (Delta(9)-THC, the active ingredient in marijuana). We show here that chronic, but not acute, administration of each of four drugs of abuse, cocaine, morphine, ethanol, and Delta(9)-THC, robustly induces DeltaFosB in nucleus accumbens, although different patterns in the core vs. shell subregions of this nucleus were apparent for the different drugs. The drugs also differed in their degree of DeltaFosB induction in dorsal striatum. In addition, all four drugs induced DeltaFosB in prefrontal cortex, with the greatest effects observed with cocaine and ethanol, and all of the drugs induced DeltaFosB to a small extent in amygdala. Furthermore, all drugs induced DeltaFosB in the hippocampus, and, with the exception of ethanol, most of this induction was seen in the dentate. Lower levels of DeltaFosB induction were seen in other brain areas in response to a particular drug treatment. These findings provide further evidence that induction of DeltaFosB in nucleus accumbens is a common action of virtually all drugs of abuse and that, beyond nucleus accumbens, each drug induces DeltaFosB in a region-specific manner in brain.