Timothy W. Whitfield

Corticosteroid-Dependent Plasticity Mediates Compulsive Alcohol Drinking in Rats

Alcoholism is characterized by a compulsion to seek and ingest alcohol, loss of control over intake, and the emergence of a negative emotional state during abstinence. We hypothesized that sustained activation of neuroendocrine stress systems (e.g., corticosteroid release via the hypothalamic-pituitary-adrenal axis) by alcohol intoxication and withdrawal and consequent alterations in glucocorticoid receptor (GR) and mineralocorticoid receptor (MR) activation drive compulsive alcohol drinking. Our results showed that rats exposed to alcohol vapor to the point of dependence displayed increased alcohol intake, compulsive drinking measured by progressive-ratio responding, and persistent alcohol consumption despite punishment, assessed by adding quinine to the alcohol solution, compared with control rats that were not exposed to alcohol vapor. No group differences were observed in the self-administration of saccharin-sweetened water. Acute alcohol withdrawal was accompanied by downregulated GR mRNA in various stress/reward-related brain regions [i.e., prefrontal cortex, nucleus accumbens (NAc), and bed nucleus of the stria terminalis (BNST)], whereas protracted alcohol abstinence was accompanied by upregulated GR mRNA in the NAc core, ventral BNST, and central nucleus of the amygdala. No significant alterations in MR mRNA levels were found. Chronic GR antagonism with mifepristone (RU38486) prevented the escalation of alcohol intake and compulsive responding induced by chronic, intermittent alcohol vapor exposure. Chronic treatment with mifepristone also blocked escalated alcohol drinking and compulsive responding during protracted abstinence. Thus, the GR system appears to be involved in the development of alcohol dependence and may represent a potential pharmacological target for the treatment of alcoholism.

A Single Intra-PFC Infusion of BDNF Prevents Cocaine-Induced Alterations in Extracellular Glutamate within the Nucleus Accumbens

The glutamatergic pathway arising in the dorsomedial prefrontal cortex (dmPFC) and projecting to the nucleus accumbens (NAc) core is a critical component of the reward circuitry that underlies reinstatement to cocaine-seeking behavior. Brain-derived neurotrophic factor (BDNF) is expressed by and modulates PFC–NAc neurons. BDNF infusion into the dmPFC attenuates reinstatement to cocaine-seeking behavior, as well as some cocaine-induced molecular adaptations within the NAc. In the present study, it is demonstrated that a single intra-dmPFC infusion of BDNF prevents cocaine self-administration-induced reduction in basal extracellular glutamate, as well as cocaine prime-induced increases in extracellular glutamate levels within the NAc. These data suggest that intra-PFC BDNF attenuates reinstatement to cocaine-seeking behavior by normalizing cocaine-induced neuroadaptations that alter glutamate neurotransmission within the NAc.

The Suppressive Effect of an Intra-Prefrontal Cortical Infusion of BDNF on Cocaine-Seeking Is Trk Receptor and Extracellular Signal-Regulated Protein Kinase Mitogen-Activated Protein Kinase Dependent

Cocaine-mediated neuroadaptations in the prefrontal cortical-nucleus accumbens pathway underlie drug-seeking in animals with a cocaine self-administration (SA) history. Neuroplasticity in the cortico-accumbens pathway is regulated, in part, by the expression and availability of neurotrophic factors, such as BDNF. We have previously demonstrated that infusion of BDNF into the dorsomedial prefrontal cortex (dmPFC) immediately after the last of 10 cocaine SA sessions attenuates contextual, cue- and cocaine prime-induced reinstatement of cocaine-seeking (Berglind et al., 2007) and normalizes cocaine-induced disruption of glutamatergic transmission in the nucleus accumbens (Berglind et al., 2009). In the present study, the suppressive effect of intra-dmPFC BDNF on cocaine-seeking is shown to depend on Trk receptor-mediated activation of extracellular signal-regulated kinase (ERK) signaling in the dmPFC. The tyrosine kinase inhibitor, K252a, and the mitogen-activated protein/extracellular signal-regulated kinase kinase inhibitor, U0126 (1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthio]butadiene), prevented BDNFs suppressive effects on cocaine-seeking. Vehicle-infused rats with a cocaine SA history showed significant decreases in ERK and cyclic AMP response element binding protein (CREB), but not Akt, phosphorylation after the final cocaine SA session that were reversed by intra-dmPFC BDNF. Additionally, BDNFs ability to normalize cocaine-mediated decreases in ERK and CREB phosphorylation was blocked by U0126, demonstrating that ERK/MAPK activation mediated the behavioral effects. This study elucidates a mechanism whereby BDNF/TrkB (tropomyosin receptor kinase B) activates ERK-regulated CREB phosphorylation in the dmPFC to counteract the neuroadaptations induced by cocaine SA and subsequent relapse to cocaine-seeking.

Long-term antagonism of κ opioid receptors prevents escalation of and increased motivation for heroin intake.

The abuse of opioid drugs, both illicit and prescription, is a persistent problem in the United States, accounting for >1.2 million users who require treatment each year. Current treatments rely on suppressing immediate withdrawal symptoms and replacing illicit drug use with long-acting opiate drugs. However, the mechanisms that lead to preventing opiate dependence are still poorly understood. We hypothesized that κ opioid receptor (KOR) activation during chronic opioid intake contributes to negative affective states associated with withdrawal and the motivation to take increasing amounts of heroin. Using a 12 h long-access model of heroin self-administration, rats showed escalation of heroin intake over several weeks. This was prevented by a single high dose (30 mg/kg) of the long-acting KOR antagonist norbinaltorphimine (nor-BNI), paralleled by reduced motivation to respond for heroin on a progressive-ratio schedule of reinforcement, a measure of compulsive-like responding. Systemic nor-BNI also significantly decreased heroin withdrawal-associated anxiety-like behavior. Immunohistochemical analysis showed prodynorphin content increased in the nucleus accumbens core in all heroin-exposed rats, but selectively increased in the nucleus accumbens shell in long-access rats. Local infusion of nor-BNI (4 μg/side) into accumbens core altered the initial intake of heroin but not the rate of escalation, while local injection into accumbens shell selectively suppressed increases in heroin intake over time without altering initial intake. These data suggest that dynorphin activity in the nucleus accumbens mediates the increasing motivation for heroin taking and compulsive-like responding for heroin, suggesting that KOR antagonists may be promising targets for the treatment of opioid addiction.

Kappa Opioid Receptor-Mediated Dysregulation of Gamma-Aminobutyric Acidergic Transmission in the Central Amygdala in Cocaine Addiction

BACKGROUND Studies have demonstrated an enhanced dynorphin/kappa-opioid receptor (KOR) system following repeated cocaine exposure, but few reports have focused on neuroadaptations within the central amygdala (CeA). METHODS We identified KOR-related physiological changes in the CeA following escalation of cocaine self-administration in rats. We used in vitro slice electrophysiological (intracellular and whole-cell recordings) methods to assess whether differential cocaine access in either 1-hour (short access [ShA]) or 6-hour (long access [LgA]) sessions induced plasticity at CeA gamma-aminobutyric acid (GABA)ergic synapses or altered the sensitivity of these synapses to KOR agonism (U50488) or antagonism (norbinaltorphimine [norBNI]). We then determined the functional effects of CeA KOR blockade in cocaine-related behaviors. RESULTS Baseline evoked GABAergic transmission was enhanced in the CeA from ShA and LgA rats compared with cocaine-naïve rats. Acute cocaine (1 µmol/L) application significantly decreased GABA release in all groups (naïve, ShA, and LgA rats). Application of U50488 (1 µmol/L) significantly decreased GABAergic transmission in the CeA from naïve rats but increased it in LgA rats. Conversely, norBNI (200 nmol/L) significantly increased GABAergic transmission in the CeA from naïve rats but decreased it in LgA rats. Norbinaltorphimine did not alter the acute cocaine-induced inhibition of GABAergic responses. Finally, CeA microinfusion of norBNI blocked cocaine-induced locomotor sensitization and attenuated the heightened anxiety-like behavior observed during withdrawal from chronic cocaine exposure in the defensive burying paradigm. CONCLUSIONS Together these data demonstrate that CeA dynorphin/KOR systems are dysregulated following excessive cocaine exposure and suggest KOR antagonism as a viable therapeutic strategy for cocaine addiction.

κ Opioid Receptors in the Nucleus Accumbens Shell Mediate Escalation of Methamphetamine Intake

Given that the κ opioid receptor (KOR) system has been implicated in psychostimulant abuse, we evaluated whether the selective KOR antagonist norbinaltorphimine dihydrochloride (nor-BNI) would attenuate the escalation of methamphetamine (METH) intake in an extended-access self-administration model. Systemic nor-BNI decreased the escalation of intake of long-access (LgA) but not short-access (ShA) self-administration. nor-BNI also decreased elevated progressive-ratio (PR) breakpoints in rats in the LgA condition and continued to decrease intake after 17 d of abstinence, demonstrating that the effects of a nor-BNI injection are long lasting. Rats with an ShA history showed an increase in prodynorphin immunoreactivity in both the nucleus accumbens (NAc) core and shell, but LgA animals showed a selective increase in the NAc shell. Other cohorts of rats received nor-BNI directly into the NAc shell or core and entered into ShA or LgA. nor-BNI infusion in the NAc shell, but not NAc core, attenuated escalation of intake and PR responding for METH in LgA rats. These data indicate that the development and/or expression of compulsive-like responding for METH under LgA conditions depends on activation of the KOR system in the NAc shell and suggest that the dynorphin–KOR system is a central component of the neuroplasticity associated with negative reinforcement systems that drive the dark side of addiction.

Probing Active Cocaine Vaccination Performance through Catalytic and Noncatalytic Hapten Design

Presently, there are no FDA-approved medications to treat cocaine addiction. Active vaccination has emerged as one approach to intervene through the rapid sequestering of the circulating drug, thus terminating both psychoactive effects and drug toxicity. Herein, we report our efforts examining two complementary, but mechanistically distinct active vaccines, i.e., noncatalytic and catalytic, for cocaine treatment. A cocaine-like hapten GNE and a cocaine transition-state analogue GNT were used to generate the active vaccines, respectively. GNE-KLH (keyhole limpet hemocyannin) was found to elicit persistent high-titer, cocaine-specific antibodies and blunt cocaine-induced locomotor behaviors. Catalytic antibodies induced by GNT-KLH were also shown to produce potent titers and suppress locomotor response in mice; however, upon repeated cocaine challenges, the vaccines protecting effects waned. In depth kinetic analysis suggested that loss of catalytic activity was due to antibody modification by cocaine. The work provides new insights for the development of active vaccines for the treatment of cocaine abuse.

Relapse to Cocaine-seeking after Abstinence Is Regulated by cAMP-dependent Protein Kinase A in the Prefrontal Cortex

Abstinence from cocaine self‐administration (SA) is associated with neuroadaptations in the prefrontal cortex (PFC) and nucleus accumbens (NAc) that are implicated in cocaine‐induced neuronal plasticity and relapse to drug‐seeking. Alterations in cAMP‐dependent protein kinase A (PKA) signaling are prominent in medium spiny neurons in the NAc after repeated cocaine exposure but it is unknown whether similar changes occur in the PFC. Because cocaine SA induces disturbances in glutamatergic transmission in the PFC‐NAc pathway, we examined whether dysregulation of PKA‐mediated molecular targets in PFC‐NAc neurons occurs during abstinence and, if so, whether it contributes to cocaine‐seeking. We measured the phosphorylation of cAMP response element binding protein (Ser133) and GluA1 (Ser845) in the dorsomedial (dm) PFC and the presynaptic marker, synapsin I (Ser9, Ser62/67, Ser603), in the NAc after 7 days of abstinence from cocaine SA with or without cue‐induced cocaine‐seeking. We also evaluated whether infusion of the PKA inhibitor, 8‐bromo‐Rp‐cyclic adenosine 3′, 5′‐monophosphorothioate (Rp‐cAMPs), into the dmPFC after abstinence would affect cue‐induced cocaine‐seeking and PKA‐regulated phosphoprotein levels. Seven days of forced abstinence increased the phosphorylation of cAMP response element binding protein and GluA1 in the dmPFC and synapsin I (Ser9) in the NAc. Induction of these phosphoproteins was reversed by a cue‐induced relapse test of cocaine‐seeking. Bilateral intra‐dmPFC Rp‐cAMPs rescued abstinence‐elevated PKA‐mediated phosphoprotein levels in the dmPFC and NAc and suppressed cue‐induced relapse. Thus, by inhibiting abstinence‐induced PKA molecular targets, relapse reverses abstinence‐induced neuroadaptations in the dmPFC that are responsible, in part, for the expression of cue‐induced cocaine‐seeking.

Probing the Effects of Hapten Stability on Cocaine Vaccine Immunogenicity

Judicious hapten design has been shown to be of importance when trying to generate a viable vaccine against a drug of abuse. Hapten design has typically been predicated upon faithfully emulating the unique chemical architecture that each drug presents. However, the need for drug-hapten congruency may also compromise vaccine immunogenicity if the drug-hapten conjugate possesses chemical epitope instability. There has been no systematic study on the impact of hapten stability as it relates to vaccine immunogenicity. As a starting point, we have probed the stability of a series of cocaine haptens through varying several of its structural elements, including functionality at the C2-position, the nature of the linker, and its site of attachment. Accordingly, a hydrolytic stability profile of four cocaine haptens (GNNA, GNNS, GNE, and GNC) was produced, and these results were compared through each haptens immunological properties, which were generated via active vaccination. From this group of four, three of the haptens, GNE, GNNA, and GNC, were further examined in an animal behavioral model, and findings here were again measured in relationship to hapten stability. We demonstrate a corresponding relationship between the half-life of the hapten and its immunogenicity, wherein haptens presenting a fully representative cocaine framework elicited higher concentrations of cocaine-specific IgG in sera and also conferred better protection against cocaine-induced locomotor activity. Our results indicate that hapten half-life plays an important role in vaccine immunogenicity and this in turn can impact animal behavioral effects when challenged with a drug of abuse.

Virus-Mediated shRNA Knockdown of Prodynorphin in the Rat Nucleus Accumbens Attenuates Depression-Like Behavior and Cocaine Locomotor Sensitization

Dynorphins, endogenous opioid peptides that arise from the precursor protein prodynorphin (Pdyn), are hypothesized to be involved in the regulation of mood states and the neuroplasticity associated with addiction. The current study tested the hypothesis that dynorphin in the nucleus accumbens (NAcc) mediates such effects. More specifically, we examined whether knockdown of Pdyn within the NAcc in rats would alter the expression of depressive-like and anxiety-like behavior, as well as cocaine locomotor sensitization. Wistar rats were injected with adeno-associated viral (AAV) vectors encoding either a Pdyn-specific short hairpin RNA (AAV-shPdyn) or a scrambled shRNA (AAV-shScr) as control. Four weeks later, rats were tested for anxiety-like behavior in the elevated plus maze test and depressive-like behavior in the forced swim test (FST). Finally, rats received one daily injection of saline or cocaine (20 mg/kg, i.p.), followed by assessment of locomotion for 4 consecutive days. Following 3 days of abstinence, the rats completed 2 additional daily cocaine/saline locomotor trials. Pdyn knockdown in the NAcc led to a significant reduction in depressive-like behavior in the FST, but had no effect on anxiety-like behavior in the elevated plus maze. Pdyn knockdown did not alter baseline locomotor behavior, the locomotor response to acute cocaine, or the initial sensitization of the locomotor response to cocaine over the first 4 cocaine treatment days. However, following 3 days abstinence the locomotor response to the cocaine challenge returned to their original levels in the AAV-shPdyn rats while remaining heightened in the AAV-shScr rats. These results suggest that dynorphin in a very specific area of the nucleus accumbens contributes to depressive-like states and may be involved in neuroadaptations in the NAcc that contribute to the development of cocaine addiction as a persistent and lasting condition.