Persistent binding at dopamine transporters determines sustained psychostimulant effects.

Psychostimulants interacting with the dopamine transporter (DAT) can be used illicitly or for the treatment of specific neuropsychiatric disorders. However, they can also produce severe and persistent adverse events. Often, their pharmacological properties in vitro do not fully correlate to their pharmacological profile in vivo. Here, we investigated the pharmacological effects of enantiomers of pyrovalerone, α-pyrrolidinovalerophenone, and 3,4-methylenedioxypyrovalerone as compared to the traditional psychostimulants cocaine and methylphenidate, using a variety of in vitro, computational, and in vivo approaches. We found that in vitro drug-binding kinetics at DAT correlate with the time-course of in vivo psychostimulant action in mice. In particular, a slow dissociation (i.e., slow koff) of S-enantiomers of pyrovalerone analogs from DAT predicts their more persistent in vivo effects when compared to cocaine and methylphenidate. Overall, our findings highlight the critical importance of drug-binding kinetics at DAT for determining the in vivo profile of effects produced by psychostimulant drugs.

Reversing anterior insular cortex neuronal hypoexcitability attenuates compulsive behavior in adolescent rats.

Development of self-regulatory competencies during adolescence is partially dependent on normative brain maturation. Here, we report that adolescent rats as compared to adults exhibit impulsive and compulsive-like behavioral traits, the latter being associated with lower expression of mRNA levels of the immediate early gene zif268 in the anterior insula cortex (AIC). This suggests that underdeveloped AIC function in adolescent rats could contribute to an immature pattern of interoceptive cue integration in decision making and a compulsive phenotype. In support of this, we report that layer 5 pyramidal neurons in the adolescent rat AIC are hypoexcitable and receive fewer glutamatergic synaptic inputs compared to adults. Chemogenetic activation of the AIC attenuated compulsive traits in adolescent rats supporting the idea that in early stages of AIC maturity there exists a suboptimal integration of sensory and cognitive information that contributes to inflexible behaviors in specific conditions of reward availability.

Muscarinic Acetylcholine M2 Receptors Regulate Lateral Habenula Neuron Activity and Control Cocaine Seeking Behavior.

The lateral habenula (LHb) balances reward and aversion by opposing activation of brain reward nuclei and is involved the inhibition of responding for cocaine in a model of impulsive behavior. Previously, we reported that the suppression of cocaine seeking was prevented by LHb inactivation or nonselective antagonism of LHb mAChRs. Here, we investigate mAChR subtypes mediating the effects of endogenous acetylcholine in this model of impulsive drug seeking and define cellular mechanisms in which mAChRs alter LHb neuron activity. Using in vitro electrophysiology, we find that LHb neurons are depolarized or hyperpolarized by the cholinergic agonists oxotremorine-M (Oxo-M) and carbachol (CCh), and that mAChRs inhibit synaptic GABA and glutamatergic inputs to these cells similarly in male and female rats. Synaptic effects of CCh were blocked by the M2-mAChR (M2R) antagonist AFDX-116 and not by pirenzepine, an M1-mAChR (M1R) antagonist. Oxo-M-mediated depolarizing currents were also blocked by AFDX-116. Although M2R activation inhibited excitatory and inhibitory inputs to LHb neurons, the effect on excitation was greater, suggesting a shift in excitatory-inhibitory balance toward net inhibition. Activation of VTA inhibitory inputs to LHb neurons, via channelrhodopsin-2 expression, evoked IPSCs that were inhibited by M2Rs. Finally, we measured LHb-dependent operant response inhibition for cocaine and found it impaired by antagonism of M2Rs, and not M1Rs. In summary, we show that a cholinergic signal to LHb and activation of M2Rs are critical to enable inhibition of responding for cocaine, and we define cellular mechanisms through which this may occur.Significance Statement:The lateral habenula (LHb) is a brain region receiving information from brain areas involved in decision-making, and its output influences motivation, reward, and movement. This interface between thoughts, emotions, and actions is how the LHb permits adaptive behavior, and LHb dysfunction is implicated in psychiatric and drug use disorders. Silencing the LHb impairs control over cocaine seeking in rats, and mAChRs are also implicated. Here, we measured cocaine seeking while blocking different mAChRs and examined mechanisms of mAChR effects on LHb neurons. M2-mAChRs were necessary for control of cocaine seeking, and these receptors altered LHb neuron activity in several ways. Our study reveals that LHb M2-mAChRs represent a potential target for treating substance use disorders.

Striatal Rgs4 regulates feeding and susceptibility to diet-induced obesity.

Consumption of high fat, high sugar (western) diets is a major contributor to the current high levels of obesity. Here, we used a multidisciplinary approach to gain insight into the molecular mechanisms underlying susceptibility to diet-induced obesity (DIO). Using positron emission tomography (PET), we identified the dorsal striatum as the brain area most altered in DIO-susceptible rats and molecular studies within this region highlighted regulator of G-protein signaling 4 (Rgs4) within laser-capture micro-dissected striatonigral (SN) and striatopallidal (SP) medium spiny neurons (MSNs) as playing a key role. Rgs4 is a GTPase accelerating enzyme implicated in plasticity mechanisms of SP MSNs, which are known to regulate feeding and disturbances of which are associated with obesity. Compared to DIO-resistant rats, DIO-susceptible rats exhibited increased striatal Rgs4 with mRNA expression levels enriched in SP MSNs. siRNA-mediated knockdown of striatal Rgs4 in DIO-susceptible rats decreased food intake to levels comparable to DIO-resistant animals. Finally, we demonstrated that the human Rgs4 gene locus is associated with increased body weight and obesity susceptibility phenotypes, and that overweight humans exhibit increased striatal Rgs4 protein. Our findings highlight a novel role for involvement of Rgs4 in SP MSNs in feeding and DIO-susceptibility.

(-)-Phenserine and the prevention of pre-programmed cell death and neuroinflammation in mild traumatic brain injury and Alzheimer's disease challenged mice.

Mild traumatic brain injury (mTBI) is a risk factor for neurodegenerative disorders, such as Alzheimer's disease (AD) and Parkinson's disease (PD). TBI-derived neuropathologies are promoted by inflammatory processes: chronic microgliosis and release of pro-inflammatory cytokines that further promote neuronal dysfunction and loss. Herein, we evaluated the effect on pre-programmed cell death/neuroinflammation/synaptic integrity and function of (-)-Phenserine tartrate (Phen), an agent originally developed for AD. This was studied at two clinically translatable doses (2.5 and 5.0 mg/kg, BID), in a weight drop (concussive) mTBI model in wild type (WT) and AD APP/PSEN1 transgenic mice. Phen mitigated mTBI-induced cognitive impairment, assessed by Novel Object Recognition and Y-maze behavioral paradigms, in WT mice. Phen fully abated mTBI-induced neurodegeneration, evaluated by counting Fluoro-Jade C-positive (FJC+) cells, in hippocampus and cortex of WT mice. In APP/PSEN1 mice, degenerating cell counts were consistently greater across all experimental groups vs. WT mice. mTBI elevated FJC+ cell counts vs. the APP/PSEN1 control (sham) group, and Phen similarly mitigated this. Anti-inflammatory effects on microglial activation (IBA1-immunoreactivity (IR)) and the pro-inflammatory cytokine TNF-α were evaluated. mTBI increased IBA1-IR and TNF-α/IBA1 colocalization vs. sham, both in WT and APP/PSEN1 mice. Phen decreased IBA1-IR throughout hippocampi and cortices of WT mice, and in cortices of AD mice. Phen, likewise, reduced levels of IBA1/TNF-α-IR colocalization volume across all areas in WT animals, with a similar trend in APP/PSEN1 mice. Actions on astrocyte activation by mTBI were followed by evaluating GFAP, and were similarly mitigated by Phen. Synaptic density was evaluated by quantifying PSD-95+ dendritic spines and Synaptophysin (Syn)-IR. Both were significantly reduced in mTBI vs. sham in both WT and APP/PSEN1 mice. Phen fully reversed the PSD-95+ spine loss in WT and Syn-IR decrease in both WT and APP/PSEN1 mice. To associate immunohistochemical changes in synaptic markers with function, hippocampal long term potentiation (LTP) was induced in WT mice. LTP was impaired by mTBI, and this impairment was mitigated by Phen. In synopsis, clinically translatable doses of Phen ameliorated mTBI-mediated pre-programmed cell death/neuroinflammation/synaptic dysfunction in WT mice, consistent with fully mitigating mTBI-induced cognitive impairments. Phen additionally demonstrated positive actions in the more pathologic brain microenvironment of AD mice, further supporting consideration of its repurposing as a treatment for mTBI.

Cannabinoid disruption of learning mechanisms involved in reward processing.

The increasing use of cannabis, its derivatives, and synthetic cannabinoids for medicinal and recreational purposes has led to burgeoning interest in understanding the addictive potential of this class of molecules. It is estimated that ∼10% of marijuana users will eventually show signs of dependence on the drug, and the diagnosis of cannabis use disorder (CUD) is increasing in the United States. The molecule that sustains the use of cannabis is Δ9-tetrahydrocannabinol (Δ9-THC), and our knowledge of its effects, and those of other cannabinoids on brain function has expanded rapidly in the past two decades. Additionally, the identification of endogenous cannabinoid (endocannabinoid) systems in brain and their roles in physiology and behavior, demonstrate extensive involvement of these lipid signaling molecules in regulating CNS function. Here, we examine roles for endogenous cannabinoids in shaping synaptic activity in cortical and subcortical brain circuits, and we discuss mechanisms in which exogenous cannabinoids, such as Δ9-THC, interact with endocannabinoid systems to disrupt neuronal network oscillations. We then explore how perturbation of the interaction of this activity within brain reward circuits may lead to impaired learning. Finally, we propose that disruption of cellular plasticity mechanisms by exogenous cannabinoids in cortical and subcortical circuits may explain the difficulty in establishing viable cannabinoid self-administration models in animals.

New technologies for examining the role of neuronal ensembles in drug addiction and fear.

Correlational data suggest that learned associations are encoded within neuronal ensembles. However, it has been difficult to prove that neuronal ensembles mediate learned behaviours because traditional pharmacological and lesion methods, and even newer cell type-specific methods, affect both activated and non-activated neurons. In addition, previous studies on synaptic and molecular alterations induced by learning did not distinguish between behaviourally activated and non-activated neurons. Here, we describe three new approaches--Daun02 inactivation, FACS sorting of activated neurons and Fos-GFP transgenic rats--that have been used to selectively target and study activated neuronal ensembles in models of conditioned drug effects and relapse. We also describe two new tools--Fos-tTA transgenic mice and inactivation of CREB-overexpressing neurons--that have been used to study the role of neuronal ensembles in conditioned fear.

Enduring Loss of Serotonergic Control of Orbitofrontal Cortex Function Following Contingent and Noncontingent Cocaine Exposure.

Clinical descriptions of cocaine addiction include compulsive drug seeking and maladaptive decision-making despite substantial aversive consequences. Research suggests that this may result from altered orbitofrontal cortex (OFC) function and its participation in outcome-based behavior. Clinical and animal studies also implicate serotonin in the regulation of OFC function in addiction and other neuropsychiatric disorders. Here we test the hypothesis that exposure to cocaine, through self-administration (CSA) or yoked-administration (CYA), alters the regulation of OFC function by 5-HT. Using whole-cell electrophysiology in brain slices from naïve rats we find that 5-HT1A receptors generate hyperpolarizing outward currents in layer-V OFC pyramidal neurons, and that 5-HT2A receptors increase glutamate release onto these cells. Following extended withdrawal from CSA or CYA, this 5-HT regulation of OFC activity is largely lost. In-situ hybridization of 5-HT receptor transcripts reveals that 5-HT1A receptor mRNA is unaffected and 5-HT2A receptor mRNA is significantly elevated after CSA or CYA. These results demonstrate that 5-HT control of OFC neurons is disrupted for extended periods following cocaine exposure. We hypothesize that this dysregulation of 5-HT signaling leads to enduring disruptions of OFC network activity that this is involved in impaired decision-making associated with cocaine addiction.

Disruption of hippocampal synaptic transmission and long-term potentiation by psychoactive synthetic cannabinoid 'Spice' compounds: comparison with Δ9 -tetrahydrocannabinol.

There has been a marked increase in the availability of synthetic drugs designed to mimic the effects of marijuana. These cannabimimetic drugs, sold illicitly as 'Spice' and related products, are associated with serious medical complications in some users. In vitro studies suggest that synthetic cannabinoids in these preparations are potent agonists at central cannabinoid CB1 receptors (CB1Rs), but few investigations have delineated their cellular effects, particularly in comparison with the psychoactive component of marijuana, Δ9 -tetrahydrocannabinol (Δ9 -THC). We compared the ability of three widely abused synthetic cannabinoids and Δ9 -THC to alter glutamate release and long-term potentiation in the mouse hippocampus. JWH-018 was the most potent inhibitor of hippocampal synaptic transmission (EC50 ~15 nM), whereas its fluoropentyl derivative, AM2201, inhibited synaptic transmission with slightly lower potency (EC50 ~60 nM). The newer synthetic cannabinoid, XLR-11, displayed much lower potency (EC50 ~900 nM) that was similar to Δ9 -THC (EC50 ~700 nM). The effects of all compounds occurred via activation of CB1Rs, as demonstrated by reversal with the selective antagonist/inverse agonist AM251 or the neutral CB1R antagonist PIMSR1. Moreover, AM2201 was without effect in the hippocampus of transgenic mice lacking the CB1R. Hippocampal slices exposed to either synthetic cannabinoids or Δ9 -THC exhibited significantly impaired long-term potentiation (LTP). We find that, compared with Δ9 -THC, the first-generation cannabinoids found in Spice preparations display higher potency, whereas a recent synthetic cannabinoid is roughly equipotent with Δ9 -THC. The disruption of synaptic function by these synthetic cannabinoids is likely to lead to profound impairments in cognitive and behavioral function.

Norepinephrine activates dopamine D4 receptors in the rat lateral habenula.

The lateral habenula (LHb) is involved in reward and aversion and is reciprocally connected with dopamine (DA)-containing brain regions, including the ventral tegmental area (VTA). We used a multidisciplinary approach to examine the properties of DA afferents to the LHb in the rat. We find that >90% of VTA tyrosine hydroxylase (TH) neurons projecting to the LHb lack vesicular monoamine transporter 2 (VMAT2) mRNA, and there is little coexpression of TH and VMAT2 protein in this mesohabenular pathway. Consistent with this, electrical stimulation of LHb did not evoke DA-like signals, assessed with fast-scan cyclic voltammetry. However, electrophysiological currents that were inhibited by L741,742, a DA-D4-receptor antagonist, were observed in LHb neurons when DA uptake or degradation was blocked. To prevent DA activation of D4 receptors, we repeated this experiment in LHb slices from DA-depleted rats. However, this did not disrupt D4 receptor activation initiated by the dopamine transporter inhibitor, GBR12935. As the LHb is also targeted by noradrenergic afferents, we examined whether GBR12935 activation of DA-D4 receptors occurred in slices depleted of norepinephrine (NE). Unlike DA, NE depletion prevented the activation of DA-D4 receptors. Moreover, direct application of NE elicited currents in LHb neurons that were blocked by L741,742, and GBR12935 was found to be a more effective blocker of NE uptake than the NE-selective transport inhibitor nisoxetine. These findings demonstrate that NE is released in the rat LHb under basal conditions and that it activates DA-D4 receptors. Therefore, NE may be an important regulator of LHb function.

Dopamine D4 receptor excitation of lateral habenula neurons via multiple cellular mechanisms.

Glutamatergic lateral habenula (LHb) output communicates negative motivational valence to ventral tegmental area (VTA) dopamine (DA) neurons via activation of the rostromedial tegmental nucleus (RMTg). However, the LHb also receives a poorly understood DA input from the VTA, which we hypothesized constitutes an important feedback loop regulating DA responses to stimuli. Using whole-cell electrophysiology in rat brain slices, we find that DA initiates a depolarizing inward current (I(DAi)) and increases spontaneous firing in 32% of LHb neurons. I(DAi) was also observed upon application of amphetamine or the DA uptake blockers cocaine or GBR12935, indicating involvement of endogenous DA. I(DAi) was blocked by D4 receptor (D4R) antagonists (L745,870 or L741,742), and mimicked by a selective D4R agonist (A412997). I(DAi) was associated with increased whole-cell conductance and was blocked by Cs+ or a selective blocker of hyperpolarization-activated cyclic nucleotide-gated (HCN) ion channel, ZD7288. I(DAi) was also associated with a depolarizing shift in half-activation voltage for the hyperpolarization-activated cation current (Ih) mediated by HCN channels. Recordings from LHb neurons containing fluorescent retrograde tracers revealed that I(DAi) was observed only in cells projecting to the RMTg and not the VTA. In parallel with direct depolarization, DA also strongly increased synaptic glutamate release and reduced synaptic GABA release onto LHb cells. These results demonstrate that DA can excite glutamatergic LHb output to RMTg via multiple cellular mechanisms. Since the RMTg strongly inhibits midbrain DA neurons, activation of LHb output to RMTg by DA represents a negative feedback loop that may dampen DA neuron output following activation.

Cocaine drives aversive conditioning via delayed activation of dopamine-responsive habenular and midbrain pathways.

Many strong rewards, including abused drugs, also produce aversive effects that are poorly understood. For example, cocaine can produce aversive conditioning after its rewarding effects have dissipated, consistent with opponent process theory, but the neural mechanisms involved are not well known. Using electrophysiological recordings in awake rats, we found that some neurons in the lateral habenula (LHb), where activation produces aversive conditioning, exhibited biphasic responses to single doses of intravenous cocaine, with an initial inhibition followed by delayed excitation paralleling cocaine's shift from rewarding to aversive. Recordings in LHb slice preparations revealed similar cocaine-induced biphasic responses and further demonstrated that biphasic responses were mimicked by dopamine, that the inhibitory phase depended on dopamine D2-like receptors, and that the delayed excitation persisted after drug washout for prolonged durations consistent with findings in vivo. c-Fos experiments further showed that cocaine-activated LHb neurons preferentially projected to and activated neurons in the rostromedial tegmental nucleus (RMTg), a recently identified target of LHb axons that is activated by negative motivational stimuli and inhibits dopamine neurons. Finally, pharmacological excitation of the RMTg produced conditioned place aversion, whereas cocaine-induced avoidance behaviors in a runway operant paradigm were abolished by lesions of LHb efferents, lesions of the RMTg, or by optogenetic inactivation of the RMTg selectively during the period when LHb neurons are activated by cocaine. Together, these results indicate that LHb/RMTg pathways contribute critically to cocaine-induced avoidance behaviors, while also participating in reciprocally inhibitory interactions with dopamine neurons.

Altered dopamine metabolism and increased vulnerability to MPTP in mice with partial deficiency of mitochondrial complex I in dopamine neurons.

A variety of observations support the hypothesis that deficiency of complex I [reduced nicotinamide-adenine dinucleotide (NADH):ubiquinone oxidoreductase] of the mitochondrial respiratory chain plays a role in the pathophysiology of Parkinson's disease (PD). However, recent data from a study using mice with knockout of the complex I subunit NADH:ubiquinone oxidoreductase iron-sulfur protein 4 (Ndufs4) has challenged this concept as these mice show degeneration of non-dopamine neurons. In addition, primary dopamine (DA) neurons derived from such mice, reported to lack complex I activity, remain sensitive to toxins believed to act through inhibition of complex I. We tissue-specifically disrupted the Ndufs4 gene in mouse heart and found an apparent severe deficiency of complex I activity in disrupted mitochondria, whereas oxidation of substrates that result in entry of electrons at the level of complex I was only mildly reduced in intact isolated heart mitochondria. Further analyses of detergent-solubilized mitochondria showed the mutant complex I to be unstable but capable of forming supercomplexes with complex I enzyme activity. The loss of Ndufs4 thus causes only a mild complex I deficiency in vivo. We proceeded to disrupt Ndufs4 in midbrain DA neurons and found no overt neurodegeneration, no loss of striatal innervation and no symptoms of Parkinsonism in tissue-specific knockout animals. However, DA homeostasis was abnormal with impaired DA release and increased levels of DA metabolites. Furthermore, Ndufs4 DA neuron knockouts were more vulnerable to the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. Taken together, these findings lend in vivo support to the hypothesis that complex I deficiency can contribute to the pathophysiology of PD.

Pharmacological characterization of a dopamine transporter ligand that functions as a cocaine antagonist.

An N-butyl analog of benztropine, JHW007 [N-(n-butyl)-3α-[bis(4'-fluorophenyl)methoxy]-tropane], binds to dopamine transporters (DAT) but has reduced cocaine-like behavioral effects and antagonizes various effects of cocaine. The present study further examined mechanisms underlying these effects. Cocaine dose-dependently increased locomotion, whereas JHW007 was minimally effective but increased activity 24 hours after injection. JHW007 (3-10 mg/kg) dose-dependently and fully antagonized the locomotor-stimulant effects of cocaine (5-60 mg/kg), whereas N-methyl and N-allyl analogs and the dopamine (DA) uptake inhibitor GBR12909 [1-(2-[bis(4-fluorophenyl)methoxy]ethyl)-4-(3-phenylpropyl)piperazine dihydrochloride] stimulated activity and failed to antagonize effects of cocaine. JHW007 also blocked the locomotor-stimulant effects of the DAT inhibitor GBR12909 but not stimulation produced by the δ-opioid agonist SNC 80 [4-[(R)-[(2S,5R)-4-allyl-2,5-dimethylpiperazin-1-yl](3-methoxyphenyl)methyl]-N,N-diethylbenzamide], which increases activity through nondopaminergic mechanisms. JHW007 blocked locomotor-stimulant effects of cocaine in both DA D2- and CB1-receptor knockout and wild-type mice, indicating a lack of involvement of these targets. Furthermore, JHW007 blocked effects of cocaine on stereotyped rearing but enhanced stereotyped sniffing, suggesting that interference with locomotion by enhanced stereotypies is not responsible for the cocaine-antagonist effects of JHW007. Time-course data indicate that administration of JHW007 antagonized the locomotor-stimulant effects of cocaine within 10 minutes of injection, whereas occupancy at the DAT, as determined in vivo, did not reach a maximum until 4.5 hours after injection. The σ1-receptor antagonist BD 1008 [N-[2-(3,4-dichlorophenyl)ethyl]-N-methyl-2-(1-pyrrolidinyl)ethylamine dihydrobromide] blocked the locomotor-stimulant effects of cocaine. Overall, these findings suggest that JHW007 has cocaine-antagonist effects that are deviate from its DAT occupancy and that some other mechanism, possibly σ-receptor antagonist activity, may contribute to the cocaine-antagonist effect of JHW007 and like drugs.

2-isoxazol-3-phenyltropane derivatives of cocaine: molecular and atypical system effects at the dopamine transporter.

The present study examined RTI-371 [3ß-(4-methylphenyl)-2ß-[3-(4-chlorophenyl)-isoxazol-5-yl]tropane], a phenyltropane cocaine analog with effects distinct from cocaine, and assessed potential mechanisms for those effects by comparison with its constitutional isomer, RTI-336 [3ß-(4-chlorophenyl)-2ß-[3-(4-methylphenyl)-isoxazol-5-yl]tropane]. In mice, RTI-371 was less effective than cocaine and RTI-336 in stimulating locomotion, and incompletely substituted (∼60% maximum at 5 minutes or 1 hour after injection) in a cocaine (10 mg/kg i.p.)/saline discrimination procedure; RTI-336 completely substituted. In contrast to RTI-336, RTI-371 was not self-administered, and its pretreatment (1.0-10 mg/kg i.p.) dose-dependently decreased maximal cocaine self-administration more potently than food-maintained responding. RTI-336 pretreatment dose-dependently left-shifted the cocaine self-administration dose-effect curve. Both RTI-336 and RTI-371 displaced [(3)H]WIN35,428 [[(3)H](-)-3ß-(4-fluorophenyl)-tropan-2ß-carboxylic acid methyl ester tartrate] binding to striatal dopamine transporters (DATs) with Ki values of 10.8 and 7.81 nM, respectively, and had lower affinities at serotonin or norepinephrine transporters, or muscarinic and σ receptors. The relative low affinity at these sites suggests the DAT as the primary target of RTI-371 with minimal contributions from these other targets. In biochemical assays probing the outward-facing DAT conformation, both RTI-371 and RTI-336 had effects similar to cocaine, suggesting little contribution of DAT conformation to the unique pharmacology of RTI-371. The locomotor-stimulant effects of RTI-371 (3.0-30 mg/kg i.p.) were comparable in wild-type and knockout cannabinoid CB1 receptor (CB1R) mice, indicating that previously reported CB1 allosteric effects do not decrease cocaine-like effects of RTI-371. DAT occupancy in vivo was most rapid with cocaine and least with RTI-371. The slow apparent association rate may allow compensatory actions that in turn dampen cocaine-like stimulation, and give RTI-371 its unique pharmacologic profile.

Medial prefrontal cortex neuronal activation and synaptic alterations after stress-induced reinstatement of palatable food seeking: a study using c-fos-GFP transgenic female rats.

Relapse to maladaptive eating habits during dieting is often provoked by stress and there is evidence for a role of ovarian hormones in stress responses and feeding. We studied the role of these hormones in stress-induced reinstatement of food seeking and medial prefrontal cortex (mPFC) neuronal activation in c-fos-GFP transgenic female rats, which express GFP in strongly activated neurons. Food-restricted ovariectomized or sham-operated c-fos-GFP rats were trained to lever-press for palatable food pellets. Subsequently, lever-pressing was extinguished and reinstatement of food seeking and mPFC neuronal activation was assessed after injections of the pharmacological stressor yohimbine (0.5-2 mg/kg) or pellet priming (1-4 noncontingent pellets). Estrous cycle effects on reinstatement were also assessed in wild-type rats. Yohimbine- and pellet-priming-induced reinstatement was associated with Fos and GFP induction in mPFC; both reinstatement and neuronal activation were minimally affected by ovarian hormones in both c-fos-GFP and wild-type rats. c-fos-GFP transgenic rats were then used to assess glutamatergic synaptic alterations within activated GFP-positive and nonactivated GFP-negative mPFC neurons following yohimbine-induced reinstatement of food seeking. This reinstatement was associated with reduced AMPA receptor/NMDA receptor current ratios and increased paired-pulse facilitation in activated GFP-positive but not GFP-negative neurons. While ovarian hormones do not appear to play a role in stress-induced relapse of food seeking in our rat model, this reinstatement was associated with unique synaptic alterations in strongly activated mPFC neurons. Our paper introduces the c-fos-GFP transgenic rat as a new tool to study unique synaptic changes in activated neurons during behavior.

PTEN deletion enhances survival, neurite outgrowth and function of dopamine neuron grafts to MitoPark mice.

Clinical trials in Parkinson's disease have shown that transplants of embryonic mesencephalic dopamine neurons form new functional connections within the host striatum, but the therapeutic benefits have been highly variable. One obstacle has been poor survival and integration of grafted dopamine neurons. Activation of Akt, a serine/threonine kinase that promotes cell survival and growth, increases the ability of neurons to survive after injury and to regenerate lost neuronal connections. Because the lipid phosphatase, phosphatase and tensin homolog (PTEN) inhibits Akt, we generated a mouse with conditional knock-out of PTEN in dopamine neurons, leading to constitutive expression of Akt in these neurons. Ventral mesencephalic tissue from dopamine phosphatase and tensin homologue knock-out or control animals was then transplanted bilaterally into the dopamine depleted striata of MitoPark mice that express a parkinsonian phenotype because of severe respiratory chain dysfunction in dopamine neurons. After transplantation into MitoPark mice, PTEN-deficient dopamine neurons were less susceptible to cell death, and exhibited a more extensive pattern of fibre outgrowth compared to control grafts. Voltammetric measurements demonstrated that dopamine release and reuptake were significantly increased in the striata of animals receiving dopamine PTEN knock-out transplants. These animals also displayed enhanced spontaneous and drug-induced locomotor activity, relative to control transplanted MitoPark mice. Our results suggest that disinhibition of the Akt-signalling pathway may provide a valuable strategy to enhance survival, function and integration of grafted dopamine neurons within the host striatum and, more generally, to improve survival and integration of different forms of neural grafts.

Attenuated response to methamphetamine sensitization and deficits in motor learning and memory after selective deletion of ß-catenin in dopamine neurons.

In the present study, we analyzed mice with a targeted deletion of ß-catenin in DA neurons (DA-ßcat KO mice) to address the functional significance of this molecule in the shaping of synaptic responses associated with motor learning and following exposure to drugs of abuse. Relative to controls, DA-ßcat KO mice showed significant deficits in their ability to form long-term memories and displayed reduced expression of methamphetamine-induced behavioral sensitization after subsequent challenge doses with this drug, suggesting that motor learning and drug-induced learning plasticity are altered in these mice. Morphological analyses showed no changes in the number or distribution of tyrosine hydroxylase-labeled neurons in the ventral midbrain. While electrochemical measurements in the striatum determined no changes in acute DA release and uptake, a small but significant decrease in DA release was detected in mutant animals after prolonged repetitive stimulation, suggesting a possible deficit in the DA neurotransmitter vesicle reserve pool. However, electron microscopy analyses did not reveal significant differences in the content of synaptic vesicles per terminal, and striatal DA levels were unchanged in DA-ßcat KO animals. In contrast, striatal mRNA levels for several markers known to regulate synaptic plasticity and DA neurotransmission were altered in DA-ßcat KO mice. This study demonstrates that ablation of ß-catenin in DA neurons leads to alterations of motor and reward-associated memories and to adaptations of the DA neurotransmitter system and suggests that ß-catenin signaling in DA neurons is required to facilitate the synaptic remodeling underlying the consolidation of long-term memories.

A subtle structural modification of a synthetic cannabinoid receptor agonist drastically increases its efficacy at the CB1 receptor.

The emergence of synthetic cannabinoid receptor agonists (SCRAs) as illicit psychoactive substances has posed considerable public health risks that include fatalities. Many SCRAs exhibit much higher efficacy and potency, compared with the phytocannabinoid Δ9-tetrahydrocannabinol (THC), at the cannabinoid receptor 1 (CB1R), a G protein-coupled receptor involved in modulating neurotransmitter release. In this study, we investigated structure activity relationships (SAR) of aminoalkylindole SCRAs at CB1Rs, focusing on 5F-pentylindoles containing an amide linker attached to different head moieties. Using in vitro bioluminescence resonance energy transfer (BRET) assays, we identified a few of SCRAs exhibiting significantly higher efficacy in engaging the Gi protein and recruiting ß-arrestin than the reference CB1R full agonist CP55940. Importantly, adding a methyl group at the head moiety of 5F-MMB-PICA yielded 5F-MDMB-PICA, an agonist exhibiting a large increase in efficacy and potency at the CB1R. This pharmacological observation was supported by a functional assay of the effects of these SCRAs on glutamate field potentials recorded in hippocampal slices. Molecular modeling and simulations of the CB1R bound with either of the SCRAs revealed critical structural determinants contributing to the higher efficacy of 5F-MDMB-PICA, and how these subtle differences propagated to the receptor-G protein interface. Thus, we find that apparently minor structural changes in the head moiety of SCRAs can cause major changes in efficacy. Our results highlight the need for close monitoring of structural modifications of newly emerging SCRAs and their potential for toxic drug responses in humans.

Subtle Structural Modification of a Synthetic Cannabinoid Receptor Agonist Drastically Increases its Efficacy at the CB1 Receptor.

The emergence of synthetic cannabinoid receptor agonists (SCRAs) as illicit psychoactive substances has posed considerable public health risks, including fatalities. Many SCRAs exhibit much higher efficacy and potency compared with the phytocannabinoid Δ9-tetrahydrocannabinol (THC) at the cannabinoid receptor 1 (CB1R), leading to dramatic differences in signaling levels that can be toxic. In this study, we investigated the structure-activity relationships of aminoalkylindole SCRAs at CB1Rs, focusing on 5F-pentylindoles containing an amide linker attached to different head moieties. Using in vitro bioluminescence resonance energy transfer assays, we identified a few SCRAs exhibiting significantly higher efficacy in engaging the Gi protein and recruiting ß-arrestin than the reference CB1R full agonist CP55940. Importantly, the extra methyl group on the head moiety of 5F-MDMB-PICA, as compared to that of 5F-MMB-PICA, led to a large increase in efficacy and potency at the CB1R. This pharmacological observation was supported by the functional effects of these SCRAs on glutamate field potentials recorded in hippocampal slices. Molecular modeling and simulations of the CB1R models bound with both of the SCRAs revealed critical structural determinants contributing to the higher efficacy of 5F-MDMB-PICA and how these subtle differences propagated to the receptor-G protein interface. Thus, we find that apparently minor structural changes in the head moiety of SCRAs can cause major changes in efficacy. Our results highlight the need for close monitoring of the structural modifications of newly emerging SCRAs and their potential for toxic drug responses in humans.