Smoking is the largest preventable cause of death and disease in the United States. However, <5% of quit attempts are successful, underscoring the urgent need for novel therapeutics. Microglia are one untapped therapeutic target. While previous studies have shown that microglia mediate both inflammatory responses in the brain and brain plasticity, little is known regarding their role in nicotine dependence and withdrawal phenotypes. Here, we examined microglial changes in the striatum—a mesolimbic region implicated in the rewarding effects of drugs and the affective disruptions occurring during withdrawal. We show that both nicotine and withdrawal induce microglial morphological changes; however, proinflammatory effects and anxiogenic behaviors were observed only during nicotine withdrawal. Pharmacological microglial depletion during withdrawal prevented these effects. These results define differential effects of nicotine and withdrawal on inflammatory signaling in the brain, laying the groundwork for development of future smoking cessation therapeutics.

Clenbuterol is a beta2 agonist/antagonist bronchodilator, and its identification in post-race samples may lead to sanctions. The objective of this study was to develop a specific and highly sensitive serum quantitation method for clenbuterol that would allow effective regulatory control of this agent in horses. Therefore, clenbuterol-d9 was synthesized for use as an internal standard, an automated solid-phase extraction method was developed, and both were used in conjunction with a multiple reaction monitoring liquid chromatography-tandem mass spectrometry (LC-MS-MS) method to allow unequivocal identification and quantitation of clenbuterol in 2 mL of serum at concentrations as low as 10 pg/mL. Five horses were dosed with oral clenbuterol (0.8 microg/kg, BID) for 10 days, and serum was collected for 14 days thereafter. Serum clenbuterol showed mean trough concentrations of approximately 150 pg/mL. After the last dose on day 10, serum clenbuterol reached a peak of approximately 500 pg/mL and then declined with a half-life of approximately 7 h. Serum clenbuterol declined to 30 and 10 pg/mL at 48 and 72 h after dosing, respectively. By 96 h after dosing, the concentration was below 4 pg/mL, the limit of detection for this method. Compared with previous results obtained in parallel urinary experiments, the serum-based approach was more reliable and satisfactory for regulation of the use of clenbuterol. Clenbuterol (90 microg) was also administered intratracheally to five horses. Peak serum concentrations of approximately 230 pg/mL were detected 10 min after administration, dropping to approximately 50 pg/mL within 30 min and declining much more slowly thereafter. These observations suggest that intratracheal administration of clenbuterol shortly before race time can be detected with this serum test. Traditionally, equine drug testing has been dependent on urine testing because of the small volume of serum samples and the low concentrations of drugs found therein. Using LC-MS-MS testing, it is now possible to unequivocally identify and quantitate low concentrations (10 pg/mL) of drugs in serum. Based on the utility of this approach, the speed with which new tests can be developed, and the confidence with which the findings can be applied in the forensic situation, this approach offers considerable scientific and regulatory advantages over more traditional urine testing approaches.

Dopamine is critical for processing of reward and etiology of drug addiction. Astrocytes throughout the brain express dopamine receptors, but consequences of astrocytic dopamine receptor signaling are not well established. We found that extracellular dopamine triggered rapid concentration-dependent stellation of astrocytic processes that was not a result of dopamine oxidation but instead relied on both cAMP-dependent and cAMP-independent dopamine receptor signaling. This was accompanied by reduced duration and increased frequency of astrocytic Ca transients, but little effect on astrocytic voltage-gated potassium channel currents. To isolate possible mechanisms underlying these structural and functional changes, we used whole-genome RNA sequencing and found prominent dopamine-induced enrichment of genes containing the CCCTC-binding factor (CTCF) motif, suggesting involvement of chromatin restructuring in the nucleus. CTCF binding to promoter sites bidirectionally regulates gene transcription and depends on activation of poly-ADP-ribose polymerase 1 (PARP1). Accordingly, antagonism of PARP1 occluded dopamine-induced changes, whereas a PARP1 agonist facilitated dopamine-induced changes on its own. These results indicate that astrocyte response to elevated dopamine involves PARP1-mediated CTCF genomic restructuring and concerted expression of gene networks. Our findings propose epigenetic regulation of chromatin landscape as a critical factor in the rapid astrocyte response to dopamine. Although dopamine is widely recognized for its role in modulating neuronal responses both in healthy and disease states, little is known about dopamine effects at non-neuronal cells in the brain. To address this gap, we performed whole-genome sequencing of astrocytes exposed to elevated extracellular dopamine and combined it with evaluation of effects on astrocyte morphology and function. We demonstrate a temporally dynamic pattern of genomic plasticity that triggers pronounced changes in astrocyte morphology and function. We further show that this plasticity depends on activation of genes sensitive to DNA-binding protein CTCF. Our results propose that a broad pattern of astrocyte responses to dopamine specifically relies on CTCF-dependent gene networks.

Clenbuterol is a beta2 agonist/antagonist bronchodilator marketed as Ventipulmin and is the only member of this group of drugs approved by the US Food and Drug Administration (FDA) for use in horses. Clenbuterol is a class 3 drug in the Association of Racing Commissioners International (ARCI) classification system; therefore, its identification in postrace samples may lead to sanctions. Recently, the sensitivity of postrace testing for clenbuterol has been substantially increased. The objective of this study was to determine the 'detection times' for clenbuterol after administration of an oral clinical dose (0.8 g/kg, b.i.d.) of Ventipulmin syrup. Five horses received oral clenbuterol (0.8 g/kg, b.i.d.) for 10 days, and urine concentrations of clenbuterol were determined by an enhanced enzyme-linked immunoabsorbent assay (ELISA) test and gas chromatography/mass spectrometric (GC/MS) analysis by two different methods for 30 days after administration. Twenty-four hours after the last administration, urine concentrations of apparent clenbuterol, as measured by ELISA, averaged about 500 ng/mL, dropping to about 1 ng/mL by day 5 posttreatment. However, there was a later transient increase in the mean concentrations of apparent clenbuterol in urine, peaking at 7 ng/mL on day 10 postadministration. The urine samples were also analysed using mass spectral quantification of both the trimethylsilyl (TMS) and methane boronic acid (MBA) derivatives of clenbuterol. Analysis using the TMS method showed that, at 24 h after the last administration, the mean concentration of recovered clenbuterol was about 22 ng/mL. Thereafter, clenbuterol concentrations fell below the limit of detection of the TMS-method by day 5 after administration but became transiently detectable again at day 10, with a mean concentration of about 1 ng/mL. Derivatization with MBA offers significant advantages over TMS for the mass spectral detection of clenbuterol, primarily because MBA derivatization yields a high molecular weight base peak of 243 m/z, which is ideal for quantitative purposes. Therefore, mass spectral analyses of selected urine samples, including the transient peak on day 10, were repeated using MBA derivatization, and comparable results were obtained. The results show that clenbuterol was undetectable in horse urine by day 5 after administration. However, an unexpected secondary peak of clenbuterol was observed at day 10 after administration that averaged approximately 1 ng/mL. Because of this secondary peak, the detection time for clenbuterol (0.8 g/kg, b.i.d. x 10 days) is at least 11 days if the threshold for detection is set at 1 ng/mL.

Neuregulin 3 (NRG3) and ErbB4 have been linked to nicotine addiction; however, the neuronal mechanisms and behavioral consequences of NRG3-ErbB4 sensitivity to nicotine remain elusive. Recent literature suggests that relapse to smoking is due to a lack of impulsive control, which is thought to be due to altered functioning within the orbitofrontal cortex (OFC). Therefore, we examined circuitry changes within this structure following nicotine application. We report that nicotine controls synaptic plasticity in the OFC through NRG3/ErbB4-dependent regulation of GABAergic inhibition. We observed that both nicotine and NRG3 facilitated the conversion of long-term potentiation into long-term depression at cortical layer 3/5 synapses. Induction of long-term depression by nicotine relied on nicotinic receptor activation and key regulators of NRG3 signaling: (1) release of intracellular calcium, (2) activation of the BACE1 beta-secretase, and (3) ErbB4 receptor activation. Nicotine-induced synaptic plasticity was also associated with accumulation of intracellular GABA and was completely blocked by GABA/GABA antagonists. To test whether these mechanisms underlie OFC-dependent behavior, we evaluated the effects of nicotine in the go/no-go task. Nicotine-impaired stimulus discrimination in this task was rescued by pharmacologic disruption of the NRG3 receptor, ErbB4. Altogether, our data indicate that nicotine-induced synaptic plasticity in the OFC and cognitive changes depend on NRG3-ErbB4 signaling. We propose that nicotine activation of this pathway may contribute to nicotine addiction, particularly in individuals with genetic variation in NRG3.

Addiction to nicotine and the inability to quit smoking are influenced by genetic factors, emphasizing the importance of understanding how genes and drugs of abuse mechanistically impact each other. One well-characterized protein responsible for regulating both response to drugs and gene expression is the transcription factor CREB (cAMP-responsive element binding protein). Previous work indicates that hippocampal-specific alterations in CREB signaling and synaptic plasticity may underlie certain nicotine withdrawal phenotypes. However, the structure of the hippocampus possesses dorsal and ventral subregions, each differing in behavioral, anatomic and gene expression characteristics. This study examines the effects of CREB deletion specifically in the ventral or dorsal hippocampus of animals chronically treated with saline, nicotine, or undergoing 24 h withdrawal. After region-specific viral injections of AAV-GFP or AAV-CRE in CREB animals, behavioral testing measured anxiety levels, using the Novelty-Induced Hypophagia test, and cognition, using a contextual fear conditioning paradigm. Deletion of CREB in the ventral, but not dorsal, hippocampus resulted in amelioration of nicotine withdrawal-induced anxiety-like behavior in the Novelty-Induced Hypophagia test. In contrast, CREB deletion in the dorsal hippocampus resulted in learning and memory deficits in fear conditioning, whereas CREB deletion in the ventral hippocampus showed an enhancement in learning. Gene expression analysis showed differential treatment- and region-dependent alterations of several CREB target genes that are well-known markers of neuroplasticity within the hippocampus. Collectively, these data provide persuasive evidence towards the distinct roles of CREB within the dorsal and ventral hippocampus separately in mediating select nicotine withdrawal phenotypes.

Remifentanil (4-methoxycarbonyl-4-[(1-oxopropyl)phenylamino]-1-piperidinepropionic acid methyl ester) is a mu-opioid receptor agonist with considerable abuse potential in racing horses. The identification of its major equine urinary metabolite, 4-methoxycarbonyl-4-[(1-oxopropyl)phenylamino]-1-piperidinepropionic+ ++ acid, an ester hydrolysis product of remifentanil is reported. Administration of remifentanil HCl (5 mg, intravenous) produced clear-cut locomotor responses, establishing the clinical efficacy of this dose. ELISA analysis of postadministration urine samples readily detected fentanyl equivalents in these samples. Mass spectrometric analysis, using solid-phase extraction and trimethylsilyl (TMS) derivatization, showed the urine samples contained parent remifentanil in low concentrations, peaking at 1 h. More significantly, a major peak was identified as representing 4-methoxycarbonyl-4-[(1-oxopropyl)phenylamino]-1-piperidinepropionic+ ++ acid, arising from ester hydrolysis of remifentanil. This metabolite reached its maximal urinary concentrations at 1 h and was present at up to 10-fold greater concentrations than parent remifentanil. Base hydrolysis of remifentanil yielded a carboxylic acid with the same mass spectral characteristics as those of the equine metabolite. In summary, these data indicate that remifentanil administration results in the appearance of readily detectable amounts of 4-methoxycarbonyl-4-[(1-oxopropyl)phenylamino]-1-piperidinepropionic+ ++ acid in urine. On this basis, screening and confirmation tests for this equine urinary metabolite should be optimized for forensic control of remifentanil.

Lidocaine is a local anesthetic drug that is widely used in equine medicine. It has the advantage of giving good local anesthesia and a longer duration of action than procaine. Charles C. Thomas, Springfield, IL). Although approved for use in horses in training by the American Association of Equine Practitioners (AAEP), lidocaine is also an Association of Racing Commissioners International (ARCI) Class 2 drug and its detection in forensic samples can result in significant penalties. Lidocaine was observed as a monoprotonated ion at m/z 235 by ESI+ MS/MS (electrospray ionization‐positive ion mode) analysis. The base peak ion at m/z 86, representing the postulated methylenediethylamino fragment [CH2N(CH2CH3)2]+, was characteristic of lidocaine and 3‐hydroxylidocaine in both ESI+ and EI (electron impact‐positive ion mode) mass spectrometry. In addition, we identified an ion at m/z 427 as the principal parent ion of the ion at m/z 86, consistent with the presence of a protonated analog of 3‐hydroxylidocaine‐glucuronide. We also sought to establish post‐administration ELISA‐based ‘detection times’ for lidocaine and lidocaine‐related compounds in urine following single subcutaneous injections of various doses (10, 40, 400 mg). Our findings suggest relatively long ELISA based ‘detection times’ for lidocaine following higher doses of this drug.