Molecular medicine (Cambridge, Mass.), Jan 18, 2016
The cadherin 13 (CDH13) gene encodes a cell adhesion molecule likely to influence development and... more The cadherin 13 (CDH13) gene encodes a cell adhesion molecule likely to influence development and connections of brain circuits that modulate addiction, locomotion and cognition, including those that involve midbrain dopamine neurons. Human CDH13 mRNA expression differs by more than 80% in postmortem cerebral cortical samples from individuals with different CDH13 genotypes, supporting examination of mice with altered Cdh13 expression as models for common human variation at this locus. Constitutive cdh13 knockout mice display evidence for changed cocaine reward: shifted dose response relationship in tests of cocaine-conditioned place preference using doses that do not alter cocaine conditioned taste aversion. Reduced adult Cdh13 expression in conditional knockouts also alters cocaine reward in ways that correlate with individual differences in cortical Cdh13 mRNA levels. In control and comparison behavioral assessments, knockout mice display modestly-quicker acquisition of rotarod an...
Stereotypical behaviors induced by methamphetamine (METH) overdose are one of the overt symptoms ... more Stereotypical behaviors induced by methamphetamine (METH) overdose are one of the overt symptoms of METH abuse, which can be easily assessed in animal models. Currently, there is no successful treatment for METH overdose. There is increasing evidence that elevated levels of brain histamine can attenuate METH-induced behavioral abnormalities, which might therefore constitute a novel therapeutic treatment for METH abuse and METH overdose. In mammals, histamine N-methyltransferase (HMT) is the sole enzyme responsible for degrading histamine in the brain. Metoprine, one of the most potent HMT inhibitors, can cross the blood-brain barrier and increase brain histamine levels by inhibiting HMT. Consequently, this compound can be a candidate for a prototype of drugs for the treatment of METH overdose.
Aripiprazole is a third-generation atypical antipsychotic and a dopamine D2 receptor partial agon... more Aripiprazole is a third-generation atypical antipsychotic and a dopamine D2 receptor partial agonist. In the present study, we investigated whether a single administration of aripiprazole to mice, either as a pretreatment or as a posttreatment, would affect stereotypy induced by methamphetamine (METH). Pretreatment of male ICR mice with aripiprazole (1 or 10 mg/kg, i.p.) attenuated the incidence of METH-induced stereotypical behavior in a dose-dependent manner. Pretreatment of mice with 1 mg/kg aripiprazole produced an increase in the locomotor activity in mice treated with METH compared with mice treated with vehicle plus METH and with 10 mg/kg aripiprazole plus METH. This increase in locomotion is indicative of a rightward shift in the dose-response curve for METH, consistent with a shift in the type of stereotypical behavior observed from biting to sniffing. Aripiprazole posttreatment, after METH-induced stereotypical behavior, was fully expressed and also significantly attenuate...
öCocaine blocks uptake by neuronal plasma membrane transporters for dopamine, serotonin and norep... more öCocaine blocks uptake by neuronal plasma membrane transporters for dopamine, serotonin and norepinephrine, producing subjective e¡ects in humans that are both euphoric/rewarding and also fearful, jittery and aversive. Mice with gene knockouts of each of these transporters display cocaine reward, manifest by cocaine place preferences that are at least as great as wildtype values. Norepinephrine and serotonin receptor knockouts even display enhanced cocaine reward. One explanation for these observations could be that cocaine produces aversive or anhedonic e¡ects by serotonin or norepinephrine receptor blockade in wildtype mice that are removed in serotonin or norepinephrine receptor knockouts, increasing net cocaine reward. Adaptations to removing one transporter could also change the rewarding valence of blocking the remaining transporters. To test these ideas, drugs that block serotonin transporter (£uoxetine), norepinephrine transporter (nisoxetine) or all three transporters (cocaine) were examined in single-or multiple-transporter knockout mice. Fluoxetine and nisoxetine acquire rewarding properties in several knockouts that are not observed in wildtype mice. Adding serotonin transporter knockout to norepinephrine transporter knockouts dramatically potentiates cocaine reward. These and previous data provide evidence that serotonin and norepinephrine transporter blockade can contribute to the net rewarding valence of cocaine. They identify neuroadaptations that may help to explain the retention of cocaine reward by dopamine and serotonin transporter knockout mice. They are consistent with emerging hypotheses that actions at the three primary brain molecular targets for cocaine each provide distinct contributions to cocaine reward and cocaine aversion in wildtype mice, and that this balance changes in mice that develop without dopamine, norepinephrine or serotonin transporters. Published by Elsevier Science Ltd on behalf of IBRO.
Synaptic levels of the monoamine neurotransmitters dopamine, serotonin, and norepinephrine are mo... more Synaptic levels of the monoamine neurotransmitters dopamine, serotonin, and norepinephrine are modulated by their respective plasma membrane transporters, albeit with a few exceptions. Monoamine transporters remove monoamines from the synaptic cleft and thus influence the degree and duration of signaling. Abnormal concentrations of these neuronal transmitters are implicated in a number of neurological and psychiatric disorders, including addiction, depression, and attention deficit/hyperactivity disorder. This work concentrates on the norepinephrine transporter (NET), using a battery of in vivo magnetic resonance imaging techniques and histological correlates to probe the effects of genetic deletion of the norepinephrine transporter on brain metabolism, anatomy and functional connectivity. MRS recorded in the striatum of NET knockout mice indicated a lower concentration of NAA that correlates with histological observations of subtle dysmorphisms in the striatum and internal capsule. As with DAT and SERT knockout mice, we detected minimal structural alterations in NET knockout mice by tensor-based morphometric analysis. In contrast, longitudinal imaging after stereotaxic prefrontal cortical injection of manganese, an established neuronal circuitry tracer, revealed that the reward circuit in the NET knockout mouse is biased toward anterior portions of the brain. This is similar to previous results observed for the dopamine transporter (DAT) knockout mouse, but dissimilar from work with serotonin transporter (SERT) knockout mice where Mn 2+ tracings extended to more posterior structures than in wildtype animals. These observations correlate with behavioral studies indicating that SERT knockout mice display anxiety-like phenotypes, while NET knockouts and to a lesser extent DAT knockout mice display antidepressant-like phenotypic features. Thus, the mainly anterior activity detected with manganese-enhanced MRI in the DAT and NET knockout mice is likely indicative of more robust connectivity in the frontal portion of the reward circuit of the DAT and NET knockout mice compared to the SERT knockout mice.
Although cocaine is primarily known for its powerful hedonic effects, there is evidence that its ... more Although cocaine is primarily known for its powerful hedonic effects, there is evidence that its affective experience has a notable aversive component that is less well understood. A variety of pharmacological and molecular approaches have implicated enhanced monoamine (MA) neurotransmission in the aversive effects of cocaine. Although numerous studies have yielded data supportive of the role of the monoamines (indirectly and directly), the specific system suggested to be involved differs across studies and paradigms (Freeman et al., 2005b; Grupp, 1997; Roberts and Fibiger, 1997). Monoamine transporter knockout mice have been useful in the study of many different aspects of cocaine effects relevant to human drug use and addiction, yet an assessment of the effects of deletion of the genes for the dopamine, norepinephrine and serotonin transporters (DAT, NET, and SERT, respectively) on cocaine's aversive properties has yet to be performed (Uhl et al., 2002). In the current investigation, the strength of cocaine-induced aversions was compared among three groups of transgenic mice with deletions of the genes responsible for the production of one of the monoamine transporters. When compared to their respective WT controls, dopamine transporter deletion slightly attenuated cocaine-induced aversion while deletion of SERT or NET resulted in a more significant delay in the onset and strength of cocaine-induced taste aversions. The data lead us to conclude that the action of cocaine to inhibit NET contributes most substantially to its aversive effects, with some involvement of SERT and minimal contribution of DAT.
Despite the impact of cocaine's aversive effects on its abuse potential, the neurochemical basis ... more Despite the impact of cocaine's aversive effects on its abuse potential, the neurochemical basis of these aversive effects remains poorly understood. By blocking the reuptake of the monoamine neurotransmitters dopamine (DA), norepinephrine (NE) and serotonin (5-HT) into the presynaptic terminal, cocaine acts as a potent indirect agonist of each of these systems. The following studies attempted to assess the extent of monoaminergic mediation of cocaine's aversive effects using conditioned taste aversion (CTA) learning (Garcia, 1955). Specifically, Experiment 1 assessed the ability of selective monoamine transporter inhibitors, e.g., DAT (vanoxerine), NET (nisoxetine) and SERT (fluoxetine), to induce taste aversions (relative to cocaine). Only the NET inhibitor approximated the aversive strength of cocaine. Experiment 2 compared the effects of pretreatment of each of these transport inhibitors on the development of a cocaine-induced CTA. Pretreatment with nisoxetine and fluoxetine both attenuated cocaine-induced aversions in a manner comparable to that produced by cocaine itself. The DAT inhibitor was without effect. Combined, the results of these investigations indicate little or no involvement of dopaminergic systems in cocaine's aversive effects while NE appears to contribute most substantially, with a possible modulatory involvement by serotonin.
We previously identified KEPI as a morphine-regulated gene using subtractive hybridization and di... more We previously identified KEPI as a morphine-regulated gene using subtractive hybridization and differential display PCR. Upon phosphorylation by protein kinase C, KEPI becomes a powerful inhibitor of protein phosphatase 1. To gain insights into KEPI functions, we created KEPI knockout (KO) mice on mixed 129S6 × C57BL/6 genetic backgrounds. KEPI maps onto mouse chromosome 10 close to the locus that contains the μ-opioid receptor (Oprm1) and provides a major quantitative trait locus for morphine effects. Analysis of single nucleotide polymorphisms in and near the Oprm1 locus identified a doubly-recombinant mouse with C57BL/6 markers within 1 Mb on either side of the KEPI deletion. This strategy minimized the amount of 129S6 DNA surrounding the transgene and documented the C57BL/6 origin of the Oprm1 gene in this founder and its offspring. Recombinant KEPIKO mice displayed a) normal analgesic responses and normal locomotion after initial morphine treatments, b) accelerated development of tolerance to analgesic effects of morphine, c) elevated activity of protein phosphatase 1 in thalamus, d) attenuated morphine reward as assessed by conditioned place preference. These data support roles for KEPI action in adaptive responses to repeated administration of morphine that include analgesic tolerance and drug reward.
Homozygous transgenic knockout mice without-opioid receptors lack morphine-induced antinociceptio... more Homozygous transgenic knockout mice without-opioid receptors lack morphine-induced antinociception, locomotion, tolerance, physical dependence, and reward. receptors thus appear to play central roles in these morphine actions. Different levels of receptor expression are found in different humans and in different animal strains. In vitro studies indicate that some morphine responses persist after inactivation of as many as 90% of the initial receptor complement, while others are attenuated after inactivating many fewer receptors. Varying levels of receptor reserve could thus exist in different-expressing neuronal populations in vivo. Heterozygous receptor knockout mice express half of wild-type receptor levels. Tests of morphine actions in these mice reveal evidence for differing receptor reserves in brain circuits that mediate distinct opiate effects. Heterozygotes display attenuated locomotion, reduced morphine self-administration, intact tolerance, rightward shifts in morphine lethality dose/effect relationships, and variable effects on place preference compared to wild-type mice. They demonstrate full physical dependence, as measured by naloxone-precipitated abstinence following five days of morphine administration. Neuroadaptive changes in sites other than receptors could be involved in some of these results. Nevertheless, these data document substantial influences that individual differences in levels of receptor expression could exert on distinct opiate drug effects. They support the idea that functional receptor reserve differs among the diverse neuronal populations that mediate distinct properties of opiate drugs.
Cocaine conditioned place preference (CPP) is intact in dopamine transporter (DAT) knockout (KO) ... more Cocaine conditioned place preference (CPP) is intact in dopamine transporter (DAT) knockout (KO) mice and enhanced in serotonin transporter (SERT) KO mice. However, cocaine CPP is eliminated in double-KO mice with no DAT and either no or one SERT gene copy. To help determine mechanisms underlying these effects, we now report examination of baselines and drug-induced changes of extracellular dopamine (DA ex) and serotonin (5-HT ex) levels in microdialysates from nucleus accumbens (NAc), caudate putamen (CPu), and prefrontal cortex (PFc) of wild-type, homozygous DAT-or SERT-KO and heterozygous or homozygous DAT/SERT double-KO mice, which are differentially rewarded by cocaine. Cocaine fails to increase DA ex in NAc of DAT-KO mice. By contrast, systemic cocaine enhances DA ex in both CPu and PFc of DAT-KO mice though local cocaine fails to affect DA ex in CPu. Adding SERT to DAT deletion attenuates the cocaine-induced DA ex increases found in CPu, but not those found in PFc. The selective SERT blocker fluoxetine increases DA ex in CPu of DAT-KO mice, while cocaine and the selective DAT blocker GBR12909 increase 5-HT ex in CPu of SERT-KO mice. These data provide evidence that (a) cocaine increases DA ex in PFc independently of DAT and that (b), in the absence of SERT, CPu levels of 5-HT ex can be increased by blocking DAT. Cocaine-induced alterations in CPu DA levels in DAT-, SERT-, and DAT/SERT double-KO mice appear to provide better correlations with cocaine CPP than cocaine-induced DA level alterations in NAc or PFc.
A series of experiments examined the effects of 8 weeks of social isolation on spontaneous locomo... more A series of experiments examined the effects of 8 weeks of social isolation on spontaneous locomotor activity, prepulse inhibition (PPl) of the acoustic startle response, latent inhibition (LI) in a conditioned suppression paradigm, and basal and d-amphetamine stimulated dopamine (DA) release in the ventral striatum, as measured by in vivo microdialysis. Both isolation-reared animals (those isolated from the weaning age) and isolation-housed animals (those isolated as adults) were hyperactive when placed in a novel environment. Social isolation also led to deficits in PPI of the acoustic startle
Because the olfactory system plays a major role in food consumption, and because "food addiction"... more Because the olfactory system plays a major role in food consumption, and because "food addiction" and associated morbidities have reached epidemic proportions, we tested the hypothesis that dietary energy restriction can modify adverse effects of cocaine on behavior and olfactory cellular and molecular plasticity. Mice maintained on an alternate day fasting (ADF) diet exhibited increased baseline locomotion and increased cocaine-sensitized locomotion during cocaine conditioning, despite no change in cocaine conditioned place preference, compared to mice fed ad libitum. Levels of dopamine and its metabolites in the olfactory bulb (OB) were suppressed in mice on the ADF diet compared to mice on the control diet, independent of acute or chronic cocaine treatment. The expression of several enzymes involved in dopamine metabolism including tyrosine hydroxylase, monoamine oxidases A and B (MAOA), and catechol-O-methyltransferase were significantly reduced in OBs of mice on the ADF diet. Both acute and chronic administration of cocaine suppressed the production of new OB cells, and this effect of cocaine was attenuated in mice on the ADF diet. Cocaine administration to mice on the control diet resulted in up-regulation of OB genes involved in mitochondrial energy metabolism, synaptic plasticity, cellular stress responses, and calcium-and cyclic AMP-mediated signaling, whereas multiple olfactory receptor genes were down-regulated by cocaine treatment. ADF abolished many of the effects of cocaine on OB gene expression. Our findings reveal that dietary energy intake modifies the neural substrates underlying some of the behavioral and physiological responses to repeated cocaine treatment, and also suggest novel roles for the olfactory system in addiction. The data further suggest that modification of dietary energy intake could provide a novel potential approach to addiction treatments.
v7−3 (SLC6A15) is the prototype for a gene subfamily whose members have sequence homologies to cl... more v7−3 (SLC6A15) is the prototype for a gene subfamily whose members have sequence homologies to classical Na +-and Cl −-dependent neurotransmitter transporters but display unusual features that include characteristic large fourth extracellular loops. Interest in v7−3 has been increased by elucidation of its expression in neurons located in cerebral cortex, hippocampus, cerebellum, midbrain, and olfactory bulb. To help clarify the role of v7−3 in brain functions, we have created and characterized v7−3 knockout mice. These mice lack functional v7−3 protein, but are viable and fertile. While our studies were in progress, v7−3 expression was reported to confer transport of proline and branched-chain amino acids in in vitro expression systems (Takanaga et al. 2005; Broer et al. 2006). Assessment of amino acid uptake into cortical synaptosomes of v 7−3 knockouts identifies 15% and 40% reductions in sodium-dependent proline and leucine transport, respectively, compared to wild type controls. Despite these biochemical changes, v7−3 knockout mice demonstrate only modest alterations in rotarod performance with aging and lack reproducible alterations in other motor, memory, anxiety or olfactory tests. Compensation for the lack of v7−3 via other amino acid carriers is likely to leave v7−3 knockouts without gross behavioral manifestations. The current results place v7−3 in the context of other brain transporters that accumulate proline and branched-chain amino acids.
Dopamine (DA) is accumulated and compartmentalized by the dopamine transporter (DAT; SLC3A6) and ... more Dopamine (DA) is accumulated and compartmentalized by the dopamine transporter (DAT; SLC3A6) and the vesicular monoamine transporter 2 (VMAT2; SLC18A2). These transporters work at the plasma and vesicular membranes of dopaminergic neurons, respectively, and thus regulate levels of DA in neuronal compartments that include the extravesicular cytoplasmic compartment. DA in this compartment has been hypothesized to contribute to oxidative damage that can reduce the function of dopaminergic neurons in aging brains and may contribute to reductions in dopaminergic neurochemical markers, locomotor behavior and responses to dopaminergic drugs that are found in aged animals. The studies reported here examined aged mice with heterozygous deletions of VMAT2 or of DAT, which each reduce transporter expression to about 50% of levels found in wild-type (WT) mice. Aged mice displayed reduced locomotor responses under a variety of circumstances, including in response to locomotor stimulants, as well as changes in monoamine levels and metabolites in a regionally dependent manner. Several effects of aging were more pronounced in heterozygous VMAT2 knockout (KO) mice, including aging induced reductions in locomotion and reduced locomotor responses to cocaine. By contrast, some effects of aging were reduced or not observed in heterozygous DAT KO mice. These findings support the idea that altered DAT and VMAT2 expression affect age-related changes in dopaminergic function. These effects are most likely mediated by alterations in DA compartmentalization, and might be hypothesized to be more exacerbated by other factors that affect the metabolism of cytosolic DA.
Recent evidence enriches our understanding of the molecular sites of action of cocaine reward and... more Recent evidence enriches our understanding of the molecular sites of action of cocaine reward and locomotor stimulation. Dopamine transporter blockade by cocaine appears a sufficient explanation for cocaine-induced locomotion. Variation in DAT appears to cause differences in locomotion without drug stimulation. However, previously-held views that DAT blockade was the sole site for cocaine reward have been replaced by a richer picture of multitransporter involvement with the rewarding and aversive actions of cocaine. These new insights, derived from studies of knockout mice with simultaneous deletions and/or blockade of multiple transporters, provide a novel model for the rewarding action of this heavily-abused substance and implicate multiple monoamine systems in cocaine's hedonic activities.
ABSTRACT The rapid rise in prevalence of type 2 diabetes mellitus (T2DM) has been driven by chang... more ABSTRACT The rapid rise in prevalence of type 2 diabetes mellitus (T2DM) has been driven by changes in environmental factors - primarily increased caloric intake and reduced energy expenditure - resulting in reduced whole body insulin sensitivity (often termed insulin resistance). Insulin resistance has been proposed to be a major driver of progression to T2DM. However, of 38 individual susceptibility loci for T2DM recently identified by genome wide association studies, by far the majority code for proteins involved in β-cell function. In this review, we discuss the possible reasons for the paucity of insulin resistance genes and ask whether the new genetic susceptibility data should focus attention on β-cell targets in the development of therapies for T2DM.
Molecular medicine (Cambridge, Mass.), Jan 18, 2016
The cadherin 13 (CDH13) gene encodes a cell adhesion molecule likely to influence development and... more The cadherin 13 (CDH13) gene encodes a cell adhesion molecule likely to influence development and connections of brain circuits that modulate addiction, locomotion and cognition, including those that involve midbrain dopamine neurons. Human CDH13 mRNA expression differs by more than 80% in postmortem cerebral cortical samples from individuals with different CDH13 genotypes, supporting examination of mice with altered Cdh13 expression as models for common human variation at this locus. Constitutive cdh13 knockout mice display evidence for changed cocaine reward: shifted dose response relationship in tests of cocaine-conditioned place preference using doses that do not alter cocaine conditioned taste aversion. Reduced adult Cdh13 expression in conditional knockouts also alters cocaine reward in ways that correlate with individual differences in cortical Cdh13 mRNA levels. In control and comparison behavioral assessments, knockout mice display modestly-quicker acquisition of rotarod an...
Stereotypical behaviors induced by methamphetamine (METH) overdose are one of the overt symptoms ... more Stereotypical behaviors induced by methamphetamine (METH) overdose are one of the overt symptoms of METH abuse, which can be easily assessed in animal models. Currently, there is no successful treatment for METH overdose. There is increasing evidence that elevated levels of brain histamine can attenuate METH-induced behavioral abnormalities, which might therefore constitute a novel therapeutic treatment for METH abuse and METH overdose. In mammals, histamine N-methyltransferase (HMT) is the sole enzyme responsible for degrading histamine in the brain. Metoprine, one of the most potent HMT inhibitors, can cross the blood-brain barrier and increase brain histamine levels by inhibiting HMT. Consequently, this compound can be a candidate for a prototype of drugs for the treatment of METH overdose.
Aripiprazole is a third-generation atypical antipsychotic and a dopamine D2 receptor partial agon... more Aripiprazole is a third-generation atypical antipsychotic and a dopamine D2 receptor partial agonist. In the present study, we investigated whether a single administration of aripiprazole to mice, either as a pretreatment or as a posttreatment, would affect stereotypy induced by methamphetamine (METH). Pretreatment of male ICR mice with aripiprazole (1 or 10 mg/kg, i.p.) attenuated the incidence of METH-induced stereotypical behavior in a dose-dependent manner. Pretreatment of mice with 1 mg/kg aripiprazole produced an increase in the locomotor activity in mice treated with METH compared with mice treated with vehicle plus METH and with 10 mg/kg aripiprazole plus METH. This increase in locomotion is indicative of a rightward shift in the dose-response curve for METH, consistent with a shift in the type of stereotypical behavior observed from biting to sniffing. Aripiprazole posttreatment, after METH-induced stereotypical behavior, was fully expressed and also significantly attenuate...
öCocaine blocks uptake by neuronal plasma membrane transporters for dopamine, serotonin and norep... more öCocaine blocks uptake by neuronal plasma membrane transporters for dopamine, serotonin and norepinephrine, producing subjective e¡ects in humans that are both euphoric/rewarding and also fearful, jittery and aversive. Mice with gene knockouts of each of these transporters display cocaine reward, manifest by cocaine place preferences that are at least as great as wildtype values. Norepinephrine and serotonin receptor knockouts even display enhanced cocaine reward. One explanation for these observations could be that cocaine produces aversive or anhedonic e¡ects by serotonin or norepinephrine receptor blockade in wildtype mice that are removed in serotonin or norepinephrine receptor knockouts, increasing net cocaine reward. Adaptations to removing one transporter could also change the rewarding valence of blocking the remaining transporters. To test these ideas, drugs that block serotonin transporter (£uoxetine), norepinephrine transporter (nisoxetine) or all three transporters (cocaine) were examined in single-or multiple-transporter knockout mice. Fluoxetine and nisoxetine acquire rewarding properties in several knockouts that are not observed in wildtype mice. Adding serotonin transporter knockout to norepinephrine transporter knockouts dramatically potentiates cocaine reward. These and previous data provide evidence that serotonin and norepinephrine transporter blockade can contribute to the net rewarding valence of cocaine. They identify neuroadaptations that may help to explain the retention of cocaine reward by dopamine and serotonin transporter knockout mice. They are consistent with emerging hypotheses that actions at the three primary brain molecular targets for cocaine each provide distinct contributions to cocaine reward and cocaine aversion in wildtype mice, and that this balance changes in mice that develop without dopamine, norepinephrine or serotonin transporters. Published by Elsevier Science Ltd on behalf of IBRO.
Synaptic levels of the monoamine neurotransmitters dopamine, serotonin, and norepinephrine are mo... more Synaptic levels of the monoamine neurotransmitters dopamine, serotonin, and norepinephrine are modulated by their respective plasma membrane transporters, albeit with a few exceptions. Monoamine transporters remove monoamines from the synaptic cleft and thus influence the degree and duration of signaling. Abnormal concentrations of these neuronal transmitters are implicated in a number of neurological and psychiatric disorders, including addiction, depression, and attention deficit/hyperactivity disorder. This work concentrates on the norepinephrine transporter (NET), using a battery of in vivo magnetic resonance imaging techniques and histological correlates to probe the effects of genetic deletion of the norepinephrine transporter on brain metabolism, anatomy and functional connectivity. MRS recorded in the striatum of NET knockout mice indicated a lower concentration of NAA that correlates with histological observations of subtle dysmorphisms in the striatum and internal capsule. As with DAT and SERT knockout mice, we detected minimal structural alterations in NET knockout mice by tensor-based morphometric analysis. In contrast, longitudinal imaging after stereotaxic prefrontal cortical injection of manganese, an established neuronal circuitry tracer, revealed that the reward circuit in the NET knockout mouse is biased toward anterior portions of the brain. This is similar to previous results observed for the dopamine transporter (DAT) knockout mouse, but dissimilar from work with serotonin transporter (SERT) knockout mice where Mn 2+ tracings extended to more posterior structures than in wildtype animals. These observations correlate with behavioral studies indicating that SERT knockout mice display anxiety-like phenotypes, while NET knockouts and to a lesser extent DAT knockout mice display antidepressant-like phenotypic features. Thus, the mainly anterior activity detected with manganese-enhanced MRI in the DAT and NET knockout mice is likely indicative of more robust connectivity in the frontal portion of the reward circuit of the DAT and NET knockout mice compared to the SERT knockout mice.
Although cocaine is primarily known for its powerful hedonic effects, there is evidence that its ... more Although cocaine is primarily known for its powerful hedonic effects, there is evidence that its affective experience has a notable aversive component that is less well understood. A variety of pharmacological and molecular approaches have implicated enhanced monoamine (MA) neurotransmission in the aversive effects of cocaine. Although numerous studies have yielded data supportive of the role of the monoamines (indirectly and directly), the specific system suggested to be involved differs across studies and paradigms (Freeman et al., 2005b; Grupp, 1997; Roberts and Fibiger, 1997). Monoamine transporter knockout mice have been useful in the study of many different aspects of cocaine effects relevant to human drug use and addiction, yet an assessment of the effects of deletion of the genes for the dopamine, norepinephrine and serotonin transporters (DAT, NET, and SERT, respectively) on cocaine's aversive properties has yet to be performed (Uhl et al., 2002). In the current investigation, the strength of cocaine-induced aversions was compared among three groups of transgenic mice with deletions of the genes responsible for the production of one of the monoamine transporters. When compared to their respective WT controls, dopamine transporter deletion slightly attenuated cocaine-induced aversion while deletion of SERT or NET resulted in a more significant delay in the onset and strength of cocaine-induced taste aversions. The data lead us to conclude that the action of cocaine to inhibit NET contributes most substantially to its aversive effects, with some involvement of SERT and minimal contribution of DAT.
Despite the impact of cocaine's aversive effects on its abuse potential, the neurochemical basis ... more Despite the impact of cocaine's aversive effects on its abuse potential, the neurochemical basis of these aversive effects remains poorly understood. By blocking the reuptake of the monoamine neurotransmitters dopamine (DA), norepinephrine (NE) and serotonin (5-HT) into the presynaptic terminal, cocaine acts as a potent indirect agonist of each of these systems. The following studies attempted to assess the extent of monoaminergic mediation of cocaine's aversive effects using conditioned taste aversion (CTA) learning (Garcia, 1955). Specifically, Experiment 1 assessed the ability of selective monoamine transporter inhibitors, e.g., DAT (vanoxerine), NET (nisoxetine) and SERT (fluoxetine), to induce taste aversions (relative to cocaine). Only the NET inhibitor approximated the aversive strength of cocaine. Experiment 2 compared the effects of pretreatment of each of these transport inhibitors on the development of a cocaine-induced CTA. Pretreatment with nisoxetine and fluoxetine both attenuated cocaine-induced aversions in a manner comparable to that produced by cocaine itself. The DAT inhibitor was without effect. Combined, the results of these investigations indicate little or no involvement of dopaminergic systems in cocaine's aversive effects while NE appears to contribute most substantially, with a possible modulatory involvement by serotonin.
We previously identified KEPI as a morphine-regulated gene using subtractive hybridization and di... more We previously identified KEPI as a morphine-regulated gene using subtractive hybridization and differential display PCR. Upon phosphorylation by protein kinase C, KEPI becomes a powerful inhibitor of protein phosphatase 1. To gain insights into KEPI functions, we created KEPI knockout (KO) mice on mixed 129S6 × C57BL/6 genetic backgrounds. KEPI maps onto mouse chromosome 10 close to the locus that contains the μ-opioid receptor (Oprm1) and provides a major quantitative trait locus for morphine effects. Analysis of single nucleotide polymorphisms in and near the Oprm1 locus identified a doubly-recombinant mouse with C57BL/6 markers within 1 Mb on either side of the KEPI deletion. This strategy minimized the amount of 129S6 DNA surrounding the transgene and documented the C57BL/6 origin of the Oprm1 gene in this founder and its offspring. Recombinant KEPIKO mice displayed a) normal analgesic responses and normal locomotion after initial morphine treatments, b) accelerated development of tolerance to analgesic effects of morphine, c) elevated activity of protein phosphatase 1 in thalamus, d) attenuated morphine reward as assessed by conditioned place preference. These data support roles for KEPI action in adaptive responses to repeated administration of morphine that include analgesic tolerance and drug reward.
Homozygous transgenic knockout mice without-opioid receptors lack morphine-induced antinociceptio... more Homozygous transgenic knockout mice without-opioid receptors lack morphine-induced antinociception, locomotion, tolerance, physical dependence, and reward. receptors thus appear to play central roles in these morphine actions. Different levels of receptor expression are found in different humans and in different animal strains. In vitro studies indicate that some morphine responses persist after inactivation of as many as 90% of the initial receptor complement, while others are attenuated after inactivating many fewer receptors. Varying levels of receptor reserve could thus exist in different-expressing neuronal populations in vivo. Heterozygous receptor knockout mice express half of wild-type receptor levels. Tests of morphine actions in these mice reveal evidence for differing receptor reserves in brain circuits that mediate distinct opiate effects. Heterozygotes display attenuated locomotion, reduced morphine self-administration, intact tolerance, rightward shifts in morphine lethality dose/effect relationships, and variable effects on place preference compared to wild-type mice. They demonstrate full physical dependence, as measured by naloxone-precipitated abstinence following five days of morphine administration. Neuroadaptive changes in sites other than receptors could be involved in some of these results. Nevertheless, these data document substantial influences that individual differences in levels of receptor expression could exert on distinct opiate drug effects. They support the idea that functional receptor reserve differs among the diverse neuronal populations that mediate distinct properties of opiate drugs.
Cocaine conditioned place preference (CPP) is intact in dopamine transporter (DAT) knockout (KO) ... more Cocaine conditioned place preference (CPP) is intact in dopamine transporter (DAT) knockout (KO) mice and enhanced in serotonin transporter (SERT) KO mice. However, cocaine CPP is eliminated in double-KO mice with no DAT and either no or one SERT gene copy. To help determine mechanisms underlying these effects, we now report examination of baselines and drug-induced changes of extracellular dopamine (DA ex) and serotonin (5-HT ex) levels in microdialysates from nucleus accumbens (NAc), caudate putamen (CPu), and prefrontal cortex (PFc) of wild-type, homozygous DAT-or SERT-KO and heterozygous or homozygous DAT/SERT double-KO mice, which are differentially rewarded by cocaine. Cocaine fails to increase DA ex in NAc of DAT-KO mice. By contrast, systemic cocaine enhances DA ex in both CPu and PFc of DAT-KO mice though local cocaine fails to affect DA ex in CPu. Adding SERT to DAT deletion attenuates the cocaine-induced DA ex increases found in CPu, but not those found in PFc. The selective SERT blocker fluoxetine increases DA ex in CPu of DAT-KO mice, while cocaine and the selective DAT blocker GBR12909 increase 5-HT ex in CPu of SERT-KO mice. These data provide evidence that (a) cocaine increases DA ex in PFc independently of DAT and that (b), in the absence of SERT, CPu levels of 5-HT ex can be increased by blocking DAT. Cocaine-induced alterations in CPu DA levels in DAT-, SERT-, and DAT/SERT double-KO mice appear to provide better correlations with cocaine CPP than cocaine-induced DA level alterations in NAc or PFc.
A series of experiments examined the effects of 8 weeks of social isolation on spontaneous locomo... more A series of experiments examined the effects of 8 weeks of social isolation on spontaneous locomotor activity, prepulse inhibition (PPl) of the acoustic startle response, latent inhibition (LI) in a conditioned suppression paradigm, and basal and d-amphetamine stimulated dopamine (DA) release in the ventral striatum, as measured by in vivo microdialysis. Both isolation-reared animals (those isolated from the weaning age) and isolation-housed animals (those isolated as adults) were hyperactive when placed in a novel environment. Social isolation also led to deficits in PPI of the acoustic startle
Because the olfactory system plays a major role in food consumption, and because "food addiction"... more Because the olfactory system plays a major role in food consumption, and because "food addiction" and associated morbidities have reached epidemic proportions, we tested the hypothesis that dietary energy restriction can modify adverse effects of cocaine on behavior and olfactory cellular and molecular plasticity. Mice maintained on an alternate day fasting (ADF) diet exhibited increased baseline locomotion and increased cocaine-sensitized locomotion during cocaine conditioning, despite no change in cocaine conditioned place preference, compared to mice fed ad libitum. Levels of dopamine and its metabolites in the olfactory bulb (OB) were suppressed in mice on the ADF diet compared to mice on the control diet, independent of acute or chronic cocaine treatment. The expression of several enzymes involved in dopamine metabolism including tyrosine hydroxylase, monoamine oxidases A and B (MAOA), and catechol-O-methyltransferase were significantly reduced in OBs of mice on the ADF diet. Both acute and chronic administration of cocaine suppressed the production of new OB cells, and this effect of cocaine was attenuated in mice on the ADF diet. Cocaine administration to mice on the control diet resulted in up-regulation of OB genes involved in mitochondrial energy metabolism, synaptic plasticity, cellular stress responses, and calcium-and cyclic AMP-mediated signaling, whereas multiple olfactory receptor genes were down-regulated by cocaine treatment. ADF abolished many of the effects of cocaine on OB gene expression. Our findings reveal that dietary energy intake modifies the neural substrates underlying some of the behavioral and physiological responses to repeated cocaine treatment, and also suggest novel roles for the olfactory system in addiction. The data further suggest that modification of dietary energy intake could provide a novel potential approach to addiction treatments.
v7−3 (SLC6A15) is the prototype for a gene subfamily whose members have sequence homologies to cl... more v7−3 (SLC6A15) is the prototype for a gene subfamily whose members have sequence homologies to classical Na +-and Cl −-dependent neurotransmitter transporters but display unusual features that include characteristic large fourth extracellular loops. Interest in v7−3 has been increased by elucidation of its expression in neurons located in cerebral cortex, hippocampus, cerebellum, midbrain, and olfactory bulb. To help clarify the role of v7−3 in brain functions, we have created and characterized v7−3 knockout mice. These mice lack functional v7−3 protein, but are viable and fertile. While our studies were in progress, v7−3 expression was reported to confer transport of proline and branched-chain amino acids in in vitro expression systems (Takanaga et al. 2005; Broer et al. 2006). Assessment of amino acid uptake into cortical synaptosomes of v 7−3 knockouts identifies 15% and 40% reductions in sodium-dependent proline and leucine transport, respectively, compared to wild type controls. Despite these biochemical changes, v7−3 knockout mice demonstrate only modest alterations in rotarod performance with aging and lack reproducible alterations in other motor, memory, anxiety or olfactory tests. Compensation for the lack of v7−3 via other amino acid carriers is likely to leave v7−3 knockouts without gross behavioral manifestations. The current results place v7−3 in the context of other brain transporters that accumulate proline and branched-chain amino acids.
Dopamine (DA) is accumulated and compartmentalized by the dopamine transporter (DAT; SLC3A6) and ... more Dopamine (DA) is accumulated and compartmentalized by the dopamine transporter (DAT; SLC3A6) and the vesicular monoamine transporter 2 (VMAT2; SLC18A2). These transporters work at the plasma and vesicular membranes of dopaminergic neurons, respectively, and thus regulate levels of DA in neuronal compartments that include the extravesicular cytoplasmic compartment. DA in this compartment has been hypothesized to contribute to oxidative damage that can reduce the function of dopaminergic neurons in aging brains and may contribute to reductions in dopaminergic neurochemical markers, locomotor behavior and responses to dopaminergic drugs that are found in aged animals. The studies reported here examined aged mice with heterozygous deletions of VMAT2 or of DAT, which each reduce transporter expression to about 50% of levels found in wild-type (WT) mice. Aged mice displayed reduced locomotor responses under a variety of circumstances, including in response to locomotor stimulants, as well as changes in monoamine levels and metabolites in a regionally dependent manner. Several effects of aging were more pronounced in heterozygous VMAT2 knockout (KO) mice, including aging induced reductions in locomotion and reduced locomotor responses to cocaine. By contrast, some effects of aging were reduced or not observed in heterozygous DAT KO mice. These findings support the idea that altered DAT and VMAT2 expression affect age-related changes in dopaminergic function. These effects are most likely mediated by alterations in DA compartmentalization, and might be hypothesized to be more exacerbated by other factors that affect the metabolism of cytosolic DA.
Recent evidence enriches our understanding of the molecular sites of action of cocaine reward and... more Recent evidence enriches our understanding of the molecular sites of action of cocaine reward and locomotor stimulation. Dopamine transporter blockade by cocaine appears a sufficient explanation for cocaine-induced locomotion. Variation in DAT appears to cause differences in locomotion without drug stimulation. However, previously-held views that DAT blockade was the sole site for cocaine reward have been replaced by a richer picture of multitransporter involvement with the rewarding and aversive actions of cocaine. These new insights, derived from studies of knockout mice with simultaneous deletions and/or blockade of multiple transporters, provide a novel model for the rewarding action of this heavily-abused substance and implicate multiple monoamine systems in cocaine's hedonic activities.
ABSTRACT The rapid rise in prevalence of type 2 diabetes mellitus (T2DM) has been driven by chang... more ABSTRACT The rapid rise in prevalence of type 2 diabetes mellitus (T2DM) has been driven by changes in environmental factors - primarily increased caloric intake and reduced energy expenditure - resulting in reduced whole body insulin sensitivity (often termed insulin resistance). Insulin resistance has been proposed to be a major driver of progression to T2DM. However, of 38 individual susceptibility loci for T2DM recently identified by genome wide association studies, by far the majority code for proteins involved in β-cell function. In this review, we discuss the possible reasons for the paucity of insulin resistance genes and ask whether the new genetic susceptibility data should focus attention on β-cell targets in the development of therapies for T2DM.
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Papers by Scott Hall