Frank Scott Hall

COCAINE MECHANISMS: ENHANCED COCAINE, FLUOXETINE AND NISOXETINE PLACE PREFERENCES FOLLOWING MONOAMINE TRANSPORTER DELETIONS

Cocaine blocks uptake by neuronal plasma membrane transporters for dopamine, serotonin and norepinephrine, producing subjective effects 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 effects 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 (fluoxetine), 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.

Animal models of depression in dopamine, serotonin and norepinephrine transporter knockout mice: prominent effects of dopamine transporter deletions

Antidepressant drugs produce therapeutic actions and many of their side effects via blockade of the plasma membrane transporters for serotonin (SERT/SLC6A2), norepinephrine (NET/SLC6A1), and dopamine (DAT/SLC6A3). Many antidepressants block several of these transporters; some are more selective. Mouse gene knockouts of these transporters provide interesting models for possible effects of chronic antidepressant treatments. To examine the role of monoamine transporters in models of depression DAT, NET, and SERT knockout (KO) mice and wild-type littermates were studied in the forced swim test (FST), the tail suspension test, and for sucrose consumption. To dissociate general activity from potential antidepressant effects three types of behavior were assessed in the FST: immobility, climbing, and swimming. In confirmation of earlier reports, both DAT KO and NET KO mice exhibited less immobility than wild-type littermates whereas SERT KO mice did not. Effects of DAT deletion were not simply because of hyperactivity, as decreased immobility was observed in DAT+/− mice that were not hyperactive as well as in DAT−/− mice that displayed profound hyperactivity. Climbing was increased, whereas swimming was almost eliminated in DAT−/− mice, and a modest but similar effect was seen in NET KO mice, which showed a modest decrease in locomotor activity. Combined increases in climbing and decreases in immobility are characteristic of FST results in antidepressant animal models, whereas selective effects on swimming are associated with the effects of stimulant drugs. Therefore, an effect on climbing is thought to more specifically reflect antidepressant effects, as has been observed in several other proposed animal models of reduced depressive phenotypes. A similar profile was observed in the tail suspension test, where DAT, NET, and SERT knockouts were all found to reduce immobility, but much greater effects were observed in DAT KO mice. However, to further determine whether these effects of DAT KO in animal models of depression may be because of the confounding effects of hyperactivity, mice were also assessed in a sucrose consumption test. Sucrose consumption was increased in DAT KO mice consistent with reduced anhedonia, and inconsistent with competitive hyperactivity; no increases were observed in SERT KO or NET KO mice. In summary, the effects of DAT KO in animal models of depression are larger than those produced by NET or SERT KO, and unlikely to be simply the result of the confounding effects of locomotor hyperactivity; thus, these data support reevaluation of the role that DAT expression could play in depression and the potential antidepressant effects of DAT blockade.

NrCAM in addiction vulnerability: positional cloning, drug-regulation, haplotype-specific expression, and altered drug reward in knockout mice.

Several lines of evidence support roles for the cell adhesion molecule NrCAM in addictions. Fine mapping within a chromosome 7 region that contains previously linked and associated genomic markers identifies NrCAM haplotypes that are associated with substance abuse vulnerabilities in four samples of abusers and controls. Differential display identifies NrCAM as a drug regulated gene. NrCAM is expressed in neurons linked to reward and memory. NrCAM displays haplotype-specific gene expression in human post-mortem brain samples. Knockout mice display reduced opiate- and stimulant-conditioned place preferences. These observations support NrCAM as a positionally cloned and drug-regulated gene whose variants are likely to change expression and alter substance abuse vulnerabilities in human addictions and animal models of drug reward.

Congenic C57BL/6 mu opiate receptor (MOR) knockout mice: baseline and opiate effects.

Homozygous µ‐opioid receptor (MOR) knockout (KO) mice developed on a chimeric C57B6/129SV background lack morphine‐induced antinociception, locomotion and reward. Therefore it appears that MOR largely mediates these morphine actions. However, one factor that could affect the extent of knockout deficits in morphine‐induced behavior is the genetic background against which the gene deletion is expressed. To examine the effect of genetic background chimeric C57B6/129SV MOR knockout mice from the 15th generation of those developed in our laboratory were backcrossed for 10 successive generations with C57BL/6 mice, a strain which is more sensitive to many of the properties of morphine, to produce congenic MOR (conMOR) KO mice. Heterozygote conMOR KO mice display attenuated morphine locomotion and reduced morphine analgesia compared to wild‐type mice. Homozygote conMOR KO mice display baseline hyperalgesia, no morphine place preference, no morphine analgesia and no morphine locomotion. These results are not qualitatively different from those observed in the MOR KO strain with a chimeric C57B6/129SV background, and suggest that although the strain has separate influences on these functions, it does not substantially interact with deletion of the µ opiate receptor gene.

A greater role for the norepinephrine transporter than the serotonin transporter in murine nociception

Norepinephrine and serotonin involvement in nociceptive functions is supported by observations of analgesic effects of norepinephrine transporter (NET) and serotonin transporter (SERT) inhibitors such as amitriptyline. However, the relative contribution of NET and SERT to baseline nociception, as well as amitriptyline analgesia, is unclear. Amitriptyline and morphine analgesia in wild-type (WT) mice and littermates with gene knockout (KO) of SERT, NET or both transporters was conducted using the hotplate and tail-flick tests. Hypoalgesia was observed in NET KO mice, and to a lesser extent in SERT KO mice. The magnitude of this hypoalgesia in NET KO mice was so profound that it limited the assessment of drug-induced analgesia. Nonetheless, the necessary exclusion of these subjects because of profound baseline hypoalgesia strongly supports the role of norepinephrine and NET in basal nociceptive behavior while indicating a much smaller role for serotonin and SERT. To further clarify the role of NET and SERT in basal nociceptive sensitivity further experiments were conducted in SERT KO and NET KO mice across a range of temperatures. NET KO mice were again found to have pronounced thermal hypoalgesia compared to WT mice in both the hotplate and tail-flick tests, while only limited effects were observed in SERT KO mice. Furthermore, in the acetic acid writhing test of visceral nociception pronounced hypoalgesia was again found in NET KO mice, but no change in SERT KO mice. As some of these effects may have resulted from developmental consequences of NET KO, the effects of the selective NET blocker nisoxetine and the selective SERT blocker fluoxetine were also examined in WT mice: only nisoxetine produced analgesia in these mice. Collectively these data suggest that NET has a far greater role in determining baseline analgesia, and perhaps other analgesic effects, than SERT in mice.

NrCAM-regulating neural systems and addiction-related behaviors.

We have previously shown that a haplotype associated with decreased NrCAM expression in brain is protective against addiction vulnerability for polysubstance abuse in humans and that Nrcam knockout mice do not develop conditioned place preferences for morphine, cocaine or amphetamine. In order to gain insight into NrCAM involvement in addiction vulnerability, which may involve specific neural circuits underlying behavioral characteristics relevant to addiction, we evaluated several behavioral phenotypes in Nrcam knockout mice. Consistent with a potential general reduction in motivational function, Nrcam knockout mice demonstrated less curiosity for novel objects and for an unfamiliar conspecific, showed also less anxiety in the zero maze. Nrcam heterozygote knockout mice reduced alcohol preference and buried fewer marbles in home cage. These observations provide further support for a role of NrCAM in substance abuse including alcoholism vulnerability, possibly through its effects on behavioral traits that may affect addiction vulnerability, including novelty seeking, obsessive compulsion and responses to aversive or anxiety‐provoking stimuli. Additionally, in order to prove glutamate homeostasis hypothesis of addiction, we analyzed glutamatergic molecules regulated by NRCAM expression. Glutaminase appears to be involved in NrCAM‐related molecular pathway in two different tissues from human and mouse. An inhibitor of the enzyme, prolyl‐leucyl‐glycinamide, treatment produced, at least, some of the phenotypes of mice shown in alcohol preference and in anxiety‐like behavior. Thus, NrCAM could affect addiction‐related behaviors via at least partially modulation of some glutamatergic pathways and neural function in brain.

Antagonistic property of buprenorphine for putative ϵ-opioid receptor-mediated G-protein activation by β-endorphin in pons/medulla of the μ-opioid receptor knockout mouse

Abstract β-Endorphin is a non-selective opioid peptide which binds μ-, δ- and putative ϵ (β-endorphin-sensitive non-μ-, non-δ- and non-κ 1 -)-opioid receptors. We have previously reported that β-endorphin-produced G-protein activation is mediated by the stimulation of both μ- and putative ϵ-opioid receptors. The present study was designed to further characterize this putative ϵ-opioid receptor-mediated G-protein activation in the pons/medulla membrane obtained from mice lacking μ-opioid receptor, using a guanosine-5′- O -(3-[ 35 S]thio)triphosphate ([ 35 S]GTPγS)-binding assay. β-Endorphin and the μ-opioid receptor agonist [ D -Ala 2 ,N-MePhe 4 ,Gly-ol 5 ]enkephalin (DAMGO) increased the [ 35 S]GTPγS binding in a concentration-dependent manner (0.001–10 μM), and at 10 μM β-endorphin and DAMGO produced approximately 250 and 120% increases of [ 35 S]GTPγS binding in the pons/medulla membrane obtained from wild-type mice, respectively. In the pons/medulla membrane obtained from μ-opioid receptor knockout mice, β-endorphin-stimulated [ 35 S]GTPγS binding was only partially attenuated and a more than 100% increase by 10 μM β-endorphin still remained, while DAMGO failed to produce any increase in [ 35 S]GTPγS binding. The residual increase in [ 35 S]GTPγS binding by 10 μM β-endorphin in μ-opioid receptor knockout mice was partially but significantly attenuated by the putative ϵ-opioid receptor partial agonist β-endorphin (1–27), but not by the δ-opioid receptor antagonist naltrindole or the κ 1 -receptor antagonist norbinaltorphimine. Furthermore, buprenorphine significantly attenuated the residual increase in [ 35 S]GTPγS binding by 10 μM β-endorphin in μ-opioid receptor knockout mice. The present results indicate that β-endorphin activates G-protein by stimulation of putative ϵ-opioid receptors in the condition lacking the μ-opioid receptor, and buprenorphine acts as an antagonist for putative ϵ-opioid receptors in this condition.

Larger Numbers of Glial and Neuronal Cells in the Periaqueductal Gray Matter of μ-Opioid Receptor Knockout Mice

Background: μ-opioid receptor knockout (MOP-KO) mice display baseline hyperalgesia. We have recently identified changes in tissue volume in the periaqueductal gray matter (PAG) using magnetic resonance imaging voxel-based morphometry. Changes in the structure and connectivity of this region might account for some behavior phenotypes in MOP-KO mice, including hyperalgesia. Methods: Adult male MOP-KO and wild-type (WT) mice were studied. Immunohistochemistry was performed to detect microglia, astrocytes, and neurons in the PAG using specific markers: ionized calcium-binding adaptor molecule 1 (Iba-1) for microglia, glial fibrillary acidic protein (GFAP) for astrocytes, and the neuronal nuclei antigen (NeuN; product of the Rbfox3 gene) for neurons, respectively. Cell counting was performed in the four parallel longitudinal columns of the PAG (dorsomedial, dorsolateral, lateral, and ventrolateral) at three different locations from bregma (−3.5, −4.0, and −4.5 mm). Results: The quantitative analysis showed larger numbers of well-distributed Iba1-IR cells (microglia), NeuN-IR cells (neurons), and GFAP-IR areas (astrocytes) at all the anatomically distinct regions examined, namely, the dorsomedial (DM) PAG, dorsolateral (DL) PAG, lateral (L) PAG, and ventrolateral (VL) PAG, in MOP-KO mice than in control mice. Conclusions: The cellular changes in the PAG identified in this paper may underlie aspects of the behavioral alterations produced by MOP receptor deletion, and suggest that alterations in the cellular structure of the PAG may contribute to hyperalgesic states.