Christie D. Fowler

Habenular α5 nicotinic receptor subunit signalling controls nicotine intake.

Genetic variation in CHRNA5, the gene encoding the α5 nicotinic acetylcholine receptor subunit, increases vulnerability to tobacco addiction and lung cancer, but the underlying mechanisms are unknown. Here we report markedly increased nicotine intake in mice with a null mutation in Chrna5. This effect was ‘rescued’ in knockout mice by re-expressing α5 subunits in the medial habenula (MHb), and recapitulated in rats through α5 subunit knockdown in MHb. Remarkably, α5 subunit knockdown in MHb did not alter the rewarding effects of nicotine but abolished the inhibitory effects of higher nicotine doses on brain reward systems. The MHb extends projections almost exclusively to the interpeduncular nucleus (IPN). We found diminished IPN activation in response to nicotine in α5 knockout mice. Further, disruption of IPN signalling increased nicotine intake in rats. Our findings indicate that nicotine activates the habenulo-interpeduncular pathway through α5-containing nAChRs, triggering an inhibitory motivational signal that acts to limit nicotine intake.

Argonaute 2 in dopamine 2 receptor-expressing neurons regulates cocaine addiction.

Cocaine is a highly addictive drug that exerts its effects by increasing the levels of released dopamine in the striatum, followed by stable changes in gene transcription, mRNA translation, and metabolism within medium spiny neurons in the striatum. The multiple changes in gene and protein expression associated with cocaine addiction suggest the existence of a mechanism that facilitates a coordinated cellular response to cocaine. Here, we provide evidence for a key role of miRNAs in cocaine addiction. We show that Argonaute 2 (Ago2), which plays an important role in miRNA generation and execution of miRNA-mediated gene silencing, is involved in regulation of cocaine addiction. Deficiency of Ago2 in dopamine 2 receptor (Drd2)–expressing neurons greatly reduces the motivation to self-administer cocaine in mice. We identified a distinct group of miRNAs that is specifically regulated by Ago2 in the striatum. Comparison of miRNAs affected by Ago2 deficiency with miRNAs that are enriched and/or up-regulated in Drd2-neurons in response to cocaine identified a set of miRNAs that are likely to play a role in cocaine addiction.

Subtypes of nicotinic acetylcholine receptors in nicotine reward, dependence, and withdrawal: Evidence from genetically modified mice

Neuronal nicotinic acetylcholine receptors (nAChRs) can regulate the activity of many neurotransmitter pathways throughout the central nervous system and are considered to be important modulators of cognition and emotion. nAChRs are also the primary site of action in the brain for nicotine, the major addictive component of tobacco smoke. nAChRs consist of five membrane-spanning subunits (&agr; and &bgr; isoforms) that can associate in various combinations to form functional nAChR ion channels. Owing to a dearth of nAChR subtype-selective ligands, the precise subunit composition of the nAChRs that regulate the rewarding effects of nicotine and the development of nicotine dependence are unknown. The advent of mice with genetic nAChR subunit modifications, however, has provided a useful experimental approach to assess the contribution of individual subunits in vivo. Here, we review data generated from nAChR subunit knockout and genetically modified mice supporting a role for discrete nAChR subunits in nicotine reinforcement and dependence processes. Importantly, the rates of tobacco dependence are far higher in patients suffering from comorbid psychiatric illnesses compared with the general population, which may at least partly reflect disease-associated alterations in nAChR signaling. An understanding of the role of nAChRs in psychiatric disorders associated with high rates of tobacco addiction, therefore, may reveal novel insights into mechanisms of nicotine dependence. Thus, we also briefly review data generated from genetically modified mice to support a role for discrete nAChR subunits in anxiety disorders, depression, and schizophrenia.

Estrogen regulation of cell proliferation and distribution of estrogen receptor‐α in the brains of adult female prairie and meadow voles

Adult female prairie (Microtus ochrogaster) and meadow (M. pennsylvanicus) voles were compared to examine neural cell proliferation and the effects of estrogen manipulation on cell proliferation in the amygdala, ventromedial hypothalamus (VMH), and dentate gyrus of the hippocampus (DG). Unlike prior studies, our study focused on the amygdala and VMH, because they are involved in social behaviors and may underlie behavioral differences between the species. Meadow voles had a higher density of cells labeled with the cell proliferation marker 5‐bromo‐2′‐deoxyuridine (BrdU) in the amygdala and DG than did prairie voles. Treatment with estradiol benzoate (EB) for 3 days increased the density of BrdU‐labeled cells in the amygdala, particularly in the posterior cortical (pCorA) and medial (pMeA) nuclei, in meadow, but not prairie, voles. Furthermore, the majority of the BrdU‐labeled cells in the pCorA and pMeA displayed either a neuronal or a glial progenitor phenotype, but no species or treatment differences were found in the percentage of neuronal or glial progenitor cells. To understand better estrogens effects on adult neurogenesis, we also examined estrogen receptor‐α (ERα) distribution. Meadow voles had more ERα‐labeled cells in the pCorA and VMH, but not in the pMeA or DG, than did prairie voles. In addition, more than one‐half of the BrdU‐labeled cells in the amygdala of both species coexpressed ERα labeling. Together, these data indicate that estrogen alters cell proliferation in a species‐ and region‐specific manner, and some of these effects may lie in the specific localization of estrogen receptors in the adult vole brain. J. Comp. Neurol. 489:166–179, 2005.

Recent advances in understanding nicotinic receptor signaling mechanisms that regulate drug self-administration behavior

Tobacco smoking is one of the leading causes of disease and premature death in the United States. Nicotine is considered the major reinforcing component in tobacco smoke responsible for tobacco addiction. Nicotine acts in the brain through the neuronal nicotinic acetylcholine receptors (nAChRs). The predominant nAChR subtypes in mammalian brain are those containing α4 and β2 subunits. The α4β2 nAChRs, particularly those located in the mesoaccumbens dopamine pathway, play a key role in regulating the reinforcing properties of nicotine. Considering that twelve mammalian nAChR subunits have been cloned, it is likely that nAChRs containing subunits in addition to, or other than, α4 and β2 also play a role in the tobacco smoking habit. Consistent with this possibility, human genome-wide association studies have shown that genetic variation in the CHRNA5-CHRNA3-CHRNB4 gene cluster located in chromosome region 15q25, which encode the α5, α3 and β4 nAChR subunits, respectively, increases vulnerability to tobacco addiction and smoking-related diseases. Most recently, α5-containing nAChRs located in the habenulo-interpeduncular tract were shown to limit intravenous nicotine self-administration behavior in rats and mice, suggesting that deficits in α5-containing nAChR signaling in the habenulo-interpeduncular tract increases vulnerability to the motivational properties of nicotine. Finally, evidence suggests that nAChRs may also play a prominent role in controlling consumption of addictive drugs other than nicotine, including cocaine, alcohol, opiates and cannabinoids. The aim of the present review is to discuss recent preclinical findings concerning the identity of the nAChR subtypes that regulate self-administration of nicotine and other drugs of abuse.

Imaging correlates of decreased axonal Na+/K+ ATPase in chronic multiple sclerosis lesions.

Degeneration of chronically demyelinated axons is a major cause of irreversible neurological decline in the human central nervous system disease, multiple sclerosis (MS). Although the molecular mechanisms responsible for this axonal degeneration remain to be elucidated, dysfunction of axonal Na+/K+ ATPase is thought to be central. To date, however, the distribution of Na+/K+ ATPase has not been studied in MS lesions.

Expression and estrogen regulation of brain-derived neurotrophic factor gene and protein in the forebrain of female prairie voles

Brain‐derived neurotrophic factor (BDNF) has been linked to the development, differentiation, and plasticity of the central nervous system. In the present study, we first used a highly specific affinity‐purified antibody and a cRNA probe to generate a detailed mapping of BDNF immunoreactive (BDNF‐ir) staining and mRNA labeling throughout the forebrain of female prairie voles. Our data revealed that (1) BDNF‐ir cells were present essentially in the brain regions in which BDNF mRNA‐labeled cells were found; (2) BDNF‐ir fibers were distributed extensively throughout many forebrain regions; and (3) BDNF mRNA was also detected in some thalamic regions in which BDNF‐ir fibers, but not immunostained cells, were present. With few exceptions, the distribution pattern of BDNF in the vole brain generally resembled the pattern found in rats. In a second experiment, we examined the effects of estrogen on BDNF expression. Ovariectomized prairie voles that were treated with estradiol benzoate had a higher level of BDNF mRNA labeling in the dentate gyrus and CA3 region of the hippocampus, as well as in the basolateral nucleus of the amygdala, than did ovariectomized voles that were treated with vehicle. In addition, estrogen treatment increased the density of BDNF‐ir fibers in the lateral septum, dorsolateral area of the bed nucleus of the stria terminalis, and lateral habenular nucleus. These data suggest that estrogen may regulate BDNF at the level of gene and protein expression, and thus, BDNF may be in a position to mediate the effects of estrogen on the brain of the prairie vole. J. Comp. Neurol. 433:499–514, 2001.

Hypocretin-1 receptors regulate the reinforcing and reward-enhancing effects of cocaine: pharmacological and behavioral genetics evidence.

Considerable evidence suggests that transmission at hypocretin-1 (orexin-1) receptors (Hcrt-R1) plays an important role in the reinstatement of extinguished cocaine-seeking behaviors in rodents. However, far less is known about the role for hypocretin transmission in regulating ongoing cocaine-taking behavior. Here, we investigated the effects of the selective Hcrt-R1 antagonist SB-334867 on cocaine intake, as measured by intravenous (IV) cocaine self-administration in rats. The stimulatory effects of cocaine on brain reward systems contribute to the establishment and maintenance of cocaine-taking behaviors. Therefore, we also assessed the effects of SB-334867 on the reward-enhancing properties of cocaine, as measured by cocaine-induced lowering of intracranial self-stimulation (ICSS) thresholds. Finally, to definitively establish a role for Hcrt-R1 in regulating cocaine intake, we assessed IV cocaine self-administration in Hcrt-R1 knockout mice. We found that SB-334867 (1–4 mg/kg) dose-dependently decreased cocaine (0.5 mg/kg/infusion) self-administration in rats but did not alter responding for food rewards under the same schedule of reinforcement. This suggests that SB-334867 decreased cocaine reinforcement without negatively impacting operant performance. SB-334867 (1–4 mg/kg) also dose-dependently attenuated the stimulatory effects of cocaine (10 mg/kg) on brain reward systems, as measured by reversal of cocaine-induced lowering of ICSS thresholds in rats. Finally, we found that Hcrt-R1 knockout mice self-administered far less cocaine than wildtype mice across the entire dose-response function. These data demonstrate that Hcrt-R1 play an important role in regulating the reinforcing and reward-enhancing properties of cocaine and suggest that hypocretin transmission is likely essential for establishing and maintaining the cocaine habit in human addicts.

Targeted Deletion of the Mouse α2 Nicotinic Acetylcholine Receptor Subunit Gene (Chrna2) Potentiates Nicotine-Modulated Behaviors

Baseline and nicotine-modulated behaviors were assessed in mice harboring a null mutant allele of the nicotinic acetylcholine receptor (nAChR) subunit gene α2 (Chrna2). Homozygous Chrna2−/− mice are viable, show expected sex and Mendelian genotype ratios, and exhibit no gross neuroanatomical abnormalities. A broad range of behavioral tests designed to assess genotype-dependent effects on anxiety (elevated plus maze and light/dark box), motor coordination (narrow bean traverse and gait), and locomotor activity revealed no significant differences between mutant mice and age-matched wild-type littermates. Furthermore, a panel of tests measuring traits, such as body position, spontaneous activity, respiration, tremors, body tone, and startle response, revealed normal responses for Chrna2-null mutant mice. However, Chrna2−/− mice do exhibit a mild motor or coordination phenotype (a decreased latency to fall during the accelerating rotarod test) and possess an increased sensitivity to nicotine-induced analgesia in the hotplate assay. Relative to wild-type, Chrna2−/− mice show potentiated nicotine self-administration and withdrawal behaviors and exhibit a sex-dependent enhancement of nicotine-facilitated cued, but not trace or contextual, fear conditioning. Overall, our results suggest that loss of the mouse nAChR α2 subunit has very limited effects on baseline behavior but does lead to the potentiation of several nicotine-modulated behaviors.

Habenular signaling in nicotine reinforcement.

Tobacco dependence is a complex genetic trait, with greater than 50% of the risk of developing dependence attributable to genetic factors (Li et al, 2003). Nicotine, the major psychoactive component in tobacco smoke responsible for dependence, functions in the brain through neuronal nicotinic acetylcholine receptors (nAChRs). A major breakthrough in understanding the genetics of tobacco dependence was the finding that allelic variation in the CHRNA3-CHRNA5-CHRNB4 gene cluster, which encodes the α3, α5, and β4 nAChR subunits, respectively, increases vulnerability to tobacco dependence and smoking-associated diseases (Bierut et al, 2008; Thorgeirsson et al, 2008). In particular, a polymorphism in CHRNA5 (rs16969968) that results in an aspartic acid to asparagine substitution at amino-acid reside 398 (D398N) more than doubles the risk of tobacco dependence in those carrying two copies of the risk allele. Little was known about how nAChRs-containing α5, α3, and/or β4 subunits may contribute to tobacco dependence. However, recent findings suggest that nAChRs containing these subunits play a key role in regulating nicotine reinforcement. The α3, α5, and β4 nAChR subunits are densely expressed in the medial habenula (MHb) and its major site of projection, the interpeduncular nucleus (IPN) (Salas et al, 2009). Our laboratory has recently shown that mice with null mutation in the α5 nAChR subunit gene intravenously self-administer significantly greater quantities of nicotine than their wild-type counterparts, particularly when higher unit doses of the drug are available for consumption (Fowler et al, 2011). This enhanced intake in the mutant mice was ameliorated by virus-mediated re-expression of α5 nAChR subunits in the MHb–IPN tract. In addition, we found that IPN neurons were insensitive to nicotine in the mutant mice, reflected in greatly diminished induction of Fos immunoreactivity in response to nicotine injections. Moreover, lidocaine-induced inactivation of the MHb or IPN increased nicotine self-administration in rats, particularly at higher units doses of the drug. Finally, virus-mediated knockdown of α5 nAChR subunits in the MHb–IPN tract did not alter the reward-enhancing properties of lower nicotine doses, but greatly attenuated the reward-inhibiting (ie, aversive) effects of higher nicotine doses in rats (Fowler et al, 2011). In keeping with these findings, overexpression of β4 nAChR subunits in the MHb–IPN tract enhanced aversion to nicotine and reduced consumption of the drug in mice (Frahm et al, 2011). Moreover, virus-mediated expression in the MHb of a major risk allele of the α5 subunit gene (D398N allele), which decreases the function of α5-containing nAChRs incorporating this risk allele and increases vulnerability to tobacco dependence in humans, reduced aversion to nicotine and enhanced nicotine intake in the β4 subunit-overexpressing mice (Frahm et al, 2011). Hence, nAChRs-containing α5 and/or β4 subunits regulate the activation of the MHb–IPN tract in response to nicotine, which signals aversion to the drug. Deficient nAChR signaling in the MHb–IPN tract, which likely occurs in humans carrying risk alleles in the CHRNA3-CHRNA5-CHRNB4 gene cluster, reduces nicotine aversion and results in greater consumption of the drug. As such, these finding reveal fundamental new insights into the mechanisms of nicotine reinforcement and the neurocircuitry of tobacco dependence.