Inhibiting cyclin-dependent kinase 5 in the nucleus accumbens enhances the expression of amphetamine-induced locomotor conditioning

When psychostimulant drugs like amphetamine are administered repeatedly in the presence of a contextual stimulus complex, long-lasting associations form between the unconditioned effects of the drug and the contextual stimuli. Here we assessed the role played by the proline-directed serine/threonine kinase cyclin-dependent kinase 5 (Cdk5) in the nucleus accumbens (NAcc) on the expression of the conditioned locomotion normally observed when rats are returned to a context previously paired with amphetamine. Infusing the Cdk5 inhibitor roscovitine (40 nmol/0.5µl/side) into the NAcc 30-min before the test for conditioning significantly enhanced the conditioned locomotor response observed in rats previously administered amphetamine in the test environment. This effect was specific to the expression of a conditioned response as inhibiting Cdk5 produced no effect in control rats previously administered saline or previously administered amphetamine elsewhere. As inhibiting Cdk5 during exposure to amphetamine has been found to block the accrual of locomotor conditioning, the present results suggest distinct roles for NAcc Cdk5 in the induction and expression of excitatory conditioning by amphetamine

Locomotor conditioning by amphetamine requires cyclin-dependent kinase 5 signaling in the nucleus accumbens

Intermittent systemic exposure to psychostimulants such as amphetamine leads to several forms of long-lasting behavioral plasticity including non-associative sensitization and associative conditioning. In the nucleus accumbens (NAcc), the protein serine/threonine kinase cyclin-dependent kinase 5 (Cdk5) and its phosphorylation target, the guanine-nucleotide exchange factor kalirin-7 (Kal7), may contribute to the neuroadaptations underlying each of these forms of plasticity. Pharmacological inhibition of Cdk5 in the NAcc prevents the increases in dendritic spine density in this site and enhances the locomotor sensitization normally observed following repeated cocaine. Mice lacking the Kal7 gene display similar phenotypes suggesting that locomotor sensitization and increased NAcc spine density need not be positively correlated. As increases in spine density may relate to the formation of associative memories and both Cdk5 and Kal7 regulate the generation of spines following repeated drug exposure, we hypothesized that either inhibiting Cdk5 or preventing its phosphorylation of Kal7 in the NAcc may prevent the induction of drug conditioning. In the present experiments, blockade in rats of NAcc Cdk5 activity with roscovitine (40 nmol/0.5µl/side) prior to each of 4 injections of amphetamine (1.5 mg/kg; i.p.) prevented the accrual of contextual locomotor conditioning but spared the induction of locomotor sensitization as revealed on tests conducted one week later. Similarly, transient viral expression in the NAcc exclusively during amphetamine exposure of a threonine-alanine mutant form of Kal7 [mKal7(T1590A)] that is not phosphorylated by Cdk5 also prevented the accrual of contextual conditioning and spared the induction of sensitization. These results indicate that Cdk5 phosphorylation of Kal7 in the NAcc is necessary for the formation of context-drug associations potentially through the modulation of dendritic spine dynamics in this site

Conjugation of haloperidol to PEG allows peripheral localisation of haloperidol and eliminates CNS extrapyramidal effects

We have previously reported the synthesis of a poly(ethylene glycol)-haloperidol (PEG-haloperidol) conjugate that retained affinity for its target D2 receptor and was stable in simulated physiological conditions. We hypothesised that this polymer-drug conjugate would localise haloperidol's activity either centrally or peripherally, dependent on the location of administration, due to the polymer preventing penetration through the blood-brain barrier (BBB). Herein, we validate this hypothesis using in vitro and in vivo studies. We first demonstrate, via a [35S]GTPγS-binding assay, that drug activity is retained after conjugation to the polymer, supportive of retention of effective therapeutic ability. Specifically, the PEG-haloperidol conjugate (at 10 and 100 nM) was able to significantly inhibit dopamine-induced G-protein activation via D2 receptors, albeit with a loss of potency compared to the free haloperidol (~18-fold at 10 nM). This loss of potency was further probed and rationalised using molecular docking experiments, which indicated that conjugated haloperidol can still bind to the D2 receptors, albeit with a flipped orientation in the binding pocket within the receptor, which may explain the reduced activity. Finally, rat catalepsy studies confirmed the restricted permeation of the conjugate through the BBB in vivo. Rats treated intravenously with free haloperidol became cataleptic, whereas normal behaviour was observed in rats that received the PEG-haloperidol conjugate, suggesting that conjugation can effectively prevent unwanted central effects. Taken together these results demonstrate that conjugating small molecules to polymers is effective at prohibiting penetration of the drug through the BBB and is a valid targeting strategy for drugs to facilitate peripheral (or central) effects without inducing side effects in other compartments

Activation of Dopamine D1-D2 Receptor Complex Attenuates Cocaine Reward and Reinstatement of Cocaine-Seeking through Inhibition of DARPP-32, ERK, and ΔFosB

A significant subpopulation of neurons in rat nucleus accumbens (NAc) coexpress dopamine D1 and D2 receptors, which can form a D1-D2 receptor complex, but their relevance in addiction is not known. The existence of the D1-D2 heteromer in the striatum of rat and monkey was established using in situ PLA, in situ FRET and co-immunoprecipitation. In rat, D1-D2 receptor heteromer activation led to place aversion and abolished cocaine CPP and locomotor sensitization, cocaine intravenous self-administration and reinstatement of cocaine seeking, as well as inhibited sucrose preference and abolished the motivation to seek palatable food. Selective disruption of this heteromer by a specific interfering peptide induced reward-like effects and enhanced the above cocaine-induced effects, including at a subthreshold dose of cocaine. The D1-D2 heteromer activated Cdk5/Thr75-DARPP-32 and attenuated cocaine-induced pERK and ΔFosB accumulation, together with inhibition of cocaine-enhanced local field potentials in NAc, blocking thus the signaling pathway activated by cocaine: D1R/cAMP/PKA/Thr34-DARPP-32/pERK with ΔFosB accumulation. In conclusion, our results show that the D1-D2 heteromer exerted tonic inhibitory control of basal natural and cocaine reward, and therefore initiates a fundamental physiologic function that limits the liability to develop cocaine addiction

Impact of stress resilience and susceptibility on fear learning, anxiety, and alcohol intake

Post-traumatic stress disorder (PTSD) can develop after exposure to traumatic events and severely impacts the quality of life. PTSD is frequently comorbid with substance use disorders, with alcoholism being particularly common. However, not everyone who experiences trauma develops PTSD and the factors that render individuals susceptible or resilient to the effects of stress are unknown although gender appears to play an important role. Rodent models of stress exposure such as stress-enhanced fear learning (SEFL) recapitulate some aspects of PTSD symptomology, making them an invaluable tool for studying this disorder. This study examined whether exposure to a modified version of the SEFL procedure (4 footshocks instead of the standard 15 over 90 min) would reveal both susceptible and resilient subjects. Following stress exposure, distinct susceptible and resilient groups emerged that differed in fear learning and anxiety-related behavior as well as voluntary alcohol intake. Some aspects of stress susceptibility manifested differently in males compared to females, with susceptibility associated with increased alcohol intake in males and increased baseline anxiety in females