Noelle C. Anastasio

Selective serotonin 5-HT2C receptor activation suppresses the reinforcing efficacy of cocaine and sucrose but differentially affects the incentive-salience value of cocaine- vs. sucrose-associated cues

Serotonin (5-HT) controls affective and motivational aspects of palatable food and drug reward and the 5-HT(2C) receptor (5-HT(2C)R) has emerged as a key regulator in this regard. We have evaluated the efficacy of a selective 5-HT(2C)R agonist, WAY 163909, in cocaine and sucrose self-administration and reinstatement assays employing parallel experimental designs in free-fed rats. WAY 163909 dose-dependently reduced the reinforcing efficacy of cocaine (ID(50) = 1.19 mg/kg) and sucrose (ID(50) = 0.7 mg/kg) as well as reinstatement (ID(50) = 0.5 mg/kg) elicited by exposure to cocaine-associated contextual cues, but not sucrose-associated contextual cues. The ID(50) of WAY 163909 predicted to decrease the reinforcing efficacy of cocaine or sucrose as well as reinstatement upon exposure to cocaine-associated cues was ∼5-12-fold lower than that predicted to suppress horizontal ambulation (ID(50) = 5.89 mg/kg) and ∼2-5-fold lower than that predicted to suppress vertical activity (ID(50) = 2.3 mg/kg). Thus, selective stimulation of the 5-HT(2C)R decreases the reinforcing efficacy of cocaine and sucrose in freely-fed rats, but differentially alters the incentive-salience value of cocaine- vs. sucrose-associated cues at doses that do not impair locomotor activity. Future research is needed to tease apart the precise contribution of 5-HT(2C)R neurocircuitry in reward and motivation and the learning and memory processes that carry the encoding for associations between environmental cues and consumption of rewarding stimuli. A more complete preclinical evaluation of these questions will ultimately allow educated proof-of-concept trials to test the efficacy of selective 5-HT(2C)R agonists as adjunctive therapy in chronic health maladies including obesity, eating disorders and drug addiction.

Brain-derived neurotrophic factor prevents phencyclidine-induced apoptosis in developing brain by parallel activation of both the ERK and PI-3K/Akt pathways.

Phencyclidine is an N-methyl d-aspartate receptor (NMDAR) blocker that has been reported to induce neuronal apoptosis during development and schizophrenia-like behaviors in rats later in life. Brain-derived neurotrophic factor (BDNF) has been shown to prevent neuronal death caused by NMDAR blockade, but the precise mechanism is unknown. This study examined the role of the phosphatidylinositol-3 kinase (PI3K)/Akt and extracellular signal-regulated kinase (ERK) pathways in BDNF protection of PCP-induced apoptosis in corticostriatal organotypic cultures. It was observed that BDNF inhibited PCP-induced apoptosis in a concentration-dependent fashion. BDNF effectively prevented PCP-induced inhibition of the ERK and PI-3K/Akt pathways and suppressed GSK-3beta activation. Blockade of either PI-3K/Akt or ERK activation abolished BDNF protection. Western blot analysis revealed that the PI-3K inhibitor LY294002 prevented the stimulating effect of BDNF on the PI-3K/Akt pathway, but had no effect on the ERK pathway. Similarly, the ERK inhibitor PD98059 prevented the stimulating effect of BDNF on the ERK pathway, but not the PI-3K/Akt pathway. Co-application of LY294002 and PD98059 had no additional effect on BDNF-evoked activation of Akt or ERK. However, concurrent exposure to PD98059 and LY294002 caused much greater inhibition of BDNF-evoked phosphorylation of GSK-3beta at serine 9 than did LY294002 alone. Finally, either BDNF or GSK-3beta inhibition prevented PCP-induced suppression of cyclic-AMP response element binding protein (CREB) phosphorylation. These data demonstrate that the protective effect of BDNF against PCP-induced apoptosis is mediated by parallel activation of the PI-3K/Akt and ERK pathways, most likely involves inhibition of GSK-3beta and activation of CREB.

Blockade of N-methyl-D-aspartate receptors by phencyclidine causes the loss of corticostriatal neurons.

Perinatal administration of the N-methyl-Dd-aspartate (NMDA) receptor antagonist phencyclidine (PCP) has been reported to produce regionally selective apoptotic cell death in the frontal cortex. The development of certain behavioral abnormalities following PCP treatment suggested that extracortical regions such as the striatum also could be affected. In this study, perinatal PCP treatment caused a marked reduction in striatal, but not hippocampal, staining for polysialic acid-neural cell adhesion molecule (PSA-NCAM), an NMDA-regulated molecule important in synaptogenesis. In order to isolate striatal influences to the cortex, this investigation was continued in vitro using corticostriatal slices. For these experiments we cultured coronal corticostriatal slices from postnatal day 7 rats. After 4 days in vitro, PCP was added for 48 h and then washed out for 24 h before harvesting the tissue. Similar to what was observed in vivo, we found that PCP treatment results in a marked reduction in striatal staining for PSA-NCAM. No change was observed in the mature form of NCAM. In striatal synaptoneurosomes, immunoblot analysis confirmed that the levels of PSA-NCAM and synaptophysin, a molecule often used as a marker of synaptogenesis, were substantially down-regulated by PCP. These effects were prevented by M40403, a superoxide dismutase mimetic that also prevented the PCP-induced terminal dUTP nick-end labeling of DNA fragments that was observed selectively in the cortex. These data suggest that PCP causes cell death by apoptosis selectively in the cortex, but not in the striatum, following either in vivo treatment of perinatal rat pups or in vitro treatment of corticostriatal slices. Further, cortical apoptosis induced by PCP negatively impacts striatal synaptogenesis, a process important in normal neural development. This deficit is probably caused by a reduction in corticostriatal neurotransmission. It is possible that the dysregulation of striatal synaptogenesis contributes to the behavioral abnormalities observed following perinatal PCP administration in vivo.

Synergism between a serotonin 5-HT2A receptor (5-HT2AR) antagonist and 5-HT2CR agonist suggests new pharmacotherapeutics for cocaine addiction.

Relapse to cocaine dependence, even after extended abstinence, involves a number of liability factors including impulsivity (predisposition toward rapid, unplanned reactions to stimuli without regard to negative consequences) and cue reactivity (sensitivity to cues associated with cocaine-taking which can promote cocaine-seeking). These factors have been mechanistically linked to serotonin (5-hydroxytryptamine, 5-HT) signaling through the 5-HT(2A) receptor (5-HT(2A)R) and 5-HT(2C)R; either a selective 5-HT(2A)R antagonist or a 5-HT(2C)R agonist suppresses impulsivity and cocaine-seeking in preclinical models. We conducted proof-of-concept analyses to evaluate whether a combination of 5-HT(2A)R antagonist plus 5-HT(2C)R agonist would have synergistic effects over these liability factors for relapse as measured in a 1-choice serial reaction time task and cocaine self-administration/reinstatement assay. Combined administration of a dose of the selective 5-HT(2A)R antagonist M100907 plus the 5-HT(2C)R agonist WAY163909, each ineffective alone, synergistically suppressed cocaine-induced hyperactivity, inherent and cocaine-evoked impulsive action, as well as cue- and cocaine-primed reinstatement of cocaine-seeking behavior. The identification of synergism between a 5-HT(2A)R antagonist plus a 5-HT(2C)R agonist to attenuate these factors important in relapse indicates the promise of a bifunctional ligand as an anti-addiction pharmacotherapeutic, setting the stage to develop new ligands with improved efficacy, potency, selectivity, and in vivo profiles over the individual molecules.

Differential regulation of the NMDA receptor by acute and sub‐chronic phencyclidine administration in the developing rat

Neurodegeneration induced by the NMDA receptor antagonist, phencyclidine (PCP), has been used to model the pathogenesis of schizophrenia in the developing rat. Acute and sub‐chronic administration of PCP in perinatal rats results in different patterns of neurodegeneration. The potential role of an alteration in the membrane expression of NMDA receptors in PCP‐induced degeneration is unknown. Acute PCP treatment on postnatal day 7 increased membrane levels of both NMDA receptor subunit 1 (NR1) and NMDA receptor subunit 2B (NR2B) proteins in the frontal cortex; conversely, NR1 and NR2B protein levels in the endoplasmic reticulum fraction were decreased. Acute PCP administration also resulted in increased membrane cortical protein levels of post‐synaptic density‐95, as well as the activation of calpain, which paralleled the observed increase in membrane expression of NR1 and NR2B. Further, administration of the calpain inhibitor, MDL28170, prevented PCP‐induced up‐regulation of NR1 and NR2B. On the other hand, sub‐chronic PCP treatment on postnatal days 7, 9 and 11 caused an increase in NR1 and NR2A expression, which was accompanied by an increase in both NR1 and NR2A in the endoplasmic reticulum fraction. Sub‐chronic PCP administration did not alter levels of post‐synaptic density‐95 and had no effect on activation of calpain. These data suggest that increased trafficking accounts for up‐regulation of cortical NR1/NR2B subunits following acute PCP administration, while increased protein synthesis likely accounts for the increased expression of NR1/NR2A following sub‐chronic PCP treatment of the developing rat. These results are discussed in the context of the differential neurodegeneration caused by acute and subchronic PCP administration in the developing rat brain.

Differential role of N-methyl-D-aspartate receptor subunits 2A and 2B in mediating phencyclidine-induced perinatal neuronal apoptosis and behavioral deficits

The mechanism underlying phencyclidine (PCP)-induced apoptosis in perinatal rats and the development of schizophrenia-like behaviors is incompletely understood. We used antagonists for N-methyl-D-aspartate (NMDA) receptor subunit NR2A- and NR2B-containing NMDA receptor to test the hypothesis that the behavioral and apoptotic effects of PCP are mediated by blockade of NR1/NR2A-containing receptors, rather than NR1/NR2B-containing receptors. Sprague-Dawley rats were treated on PN7, PN9, and PN11 with PCP (10 mg/kg), PEAQX (NR2A-preferring antagonist; 10, 20, or 40 mg/kg), or ifenprodil (selective NR2B antagonist; 1, 5, or 10 mg/kg) and sacrificed for measurement of caspase-3 activity (an index of apoptosis) or allowed to age and tested for locomotor sensitization to PCP challenge on PN28-PN35. PCP or PEAQX on PN7, PN9, and PN11 markedly elevated caspase-3 activity in the cortex; ifenprodil showed no effect. Striatal apoptosis was evident only after subchronic treatment with a high dose of PEAQX (20 mg/kg). Animals treated with PCP or PEAQX on PN7, PN9, and PN11 showed a sensitized locomotor response to PCP challenge on PN28-PN35. Ifenprodil treatment had no effect on either measure. Therefore, PCP blockade of cortical NR1/NR2A, rather than NR1/NR2B, appears to be responsible for PCP-induced apoptosis and the development of long-lasting behavioral deficits.

Serotonin 5-HT2C receptor protein expression is enriched in synaptosomal and post-synaptic compartments of rat cortex.

J. Neurochem. (2010) 113, 1504–1515.

Lithium Protection of Phencyclidine-Induced Neurotoxicity in Developing Brain: The Role of Phosphatidylinositol-3 Kinase/Akt and Mitogen-Activated Protein Kinase Kinase/Extracellular Signal-Regulated Kinase Signaling Pathways

Phencyclidine (PCP) and other N-methyl-d-aspartate (NMDA) receptor antagonists have been shown to be neurotoxic to developing brains and to result in schizophrenia-like behaviors later in development. Prevention of both effects by antischizophrenic drugs suggests the validity of PCP neurodevelopmental toxicity as a heuristic model of schizophrenia. Lithium is used for the treatment of bipolar and schizoaffective disorders and has recently been shown to have neuroprotective properties. The present study used organotypic corticostriatal slices taken from postnatal day 2 rat pups to investigate the protective effect of lithium and the role of the phosphatidylinositol-3 kinase (PI-3K)/Akt and mitogen-activated protein kinase kinase/extracellular signal-regulated kinase (MEK/ERK) pathways in PCP-induced cell death. Lithium pretreatment dose-dependently reduced PCP-induced caspase-3 activation and DNA fragmentation in layers II to IV of the cortex. PCP elicited time-dependent inhibition of the MEK/ERK and PI-3K/Akt pathways, as indicated by dephosphorylation of ERK1/2 and Akt. The proapoptotic factor glycogen synthase kinase (GSK)-3β was also dephosphorylated at serine 9 and thus activated. Lithium prevented PCP-induced inhibition of the two pathways and activation of GSK-3β. Furthermore, blocking either PI-3K/Akt or MEK/ERK pathway abolished the protective effect of lithium, whereas inhibiting GSK-3β activity mimicked the protective effect of lithium. However, no cross-talk between the two pathways was found. Finally, specific GSK-3β inhibition did not prevent PCP-induced dephosphorylation of Akt and ERK. These data strongly suggest that the protective effect of lithium against PCP-induced neuroapoptosis is mediated through independent stimulation of the PI-3K/Akt and ERK pathways and suppression of GSK-3β activity.

Functional status of the serotonin 5-HT2C receptor (5-HT2CR) drives interlocked phenotypes that precipitate relapse-like behaviors in cocaine dependence.

Relapse vulnerability in cocaine dependence is rooted in genetic and environmental determinants, and propelled by both impulsivity and the responsivity to cocaine-linked cues (‘cue reactivity’). The serotonin (5-hydroxytryptamine, 5-HT) 5-HT2C receptor (5-HT2CR) within the medial prefrontal cortex (mPFC) is uniquely poised to serve as a strategic nexus to mechanistically control these behaviors. The 5-HT2CR functional capacity is regulated by a number of factors including availability of active membrane receptor pools, the composition of the 5-HT2CR macromolecular protein complex, and editing of the 5-HT2CR pre-mRNA. The one-choice serial reaction time (1-CSRT) task was used to identify impulsive action phenotypes in an outbred rat population before cocaine self-administration and assessment of cue reactivity in the form of lever presses reinforced by the cocaine-associated discrete cue complex during forced abstinence. The 1-CSRT task reliably and reproducibly identified high impulsive (HI) and low impulsive (LI) action phenotypes; HI action predicted high cue reactivity. Lower cortical 5-HT2CR membrane protein levels concomitant with higher levels of 5-HT2CR:postsynaptic density 95 complex distinguished HI rats from LI rats. The frequency of edited 5-HT2CR mRNA variants was elevated with the prediction that the protein population in HI rats favors those isoforms linked to reduced signaling capacity. Genetic loss of the mPFC 5-HT2CR induced aggregate impulsive action/cue reactivity, suggesting that depressed cortical 5-HT2CR tone confers vulnerability to these interlocked behaviors. Thus, impulsive action and cue reactivity appear to neuromechanistically overlap in rodents, with the 5-HT2CR functional status acting as a neural rheostat to regulate, in part, the intersection between these vulnerability behaviors.

Serotonin (5-hydroxytryptamine) 5-HT2A receptor: Association with inherent and cocaine-evoked behavioral disinhibition in rats

Alterations in the balance of functional activity within the serotonin [5-hydroxytryptamine (5-HT)] system are hypothesized to underlie impulse control. Cocaine-dependent subjects consistently show greater impulsivity relative to nondrug using control subjects. Preclinical studies suggest that the 5-HT2A receptor (5-HT2AR) contributes to the regulation of impulsive behavior and also mediates some of the behavioral effects of cocaine. We hypothesized that the selective 5-HT2AR antagonist M100907 would reduce inherent levels of impulsivity and attenuate impulsive responding induced by cocaine in two animal models of impulsivity, the differential reinforcement of low rate (DRL) task and the one-choice serial reaction time (1-CSRT) task. M100907 reduced rates of responding in the DRL task and premature responding in the 1-CSRT task. Conversely, cocaine disrupted rates of responding in the DRL task and increased premature responding in the 1-CSRT task. M100907 attenuated cocaine-induced increases in specific markers of behavioral disinhibition in the DRL and 1-CSRT tasks. These results suggest that the 5-HT2AR regulates inherent impulsivity, and that blockade of the 5-HT2AR alleviates specific aspects of elevated levels of impulsivity induced by cocaine exposure. These data point to the 5-HT2AR as an important regulatory substrate in impulse control.