The real-time monitoring of specific analytes in situ in the living body would greatly advance our understanding of physiology and the development of personalized medicine. Because they are continuous (wash-free and reagentless) and are able to work in complex media (e.g., undiluted serum), electrochemical aptamer-based (E-AB) sensors are promising candidates to fill this role. E-AB sensors suffer, however, from often-severe baseline drift when deployed in undiluted whole blood either in vitro or in vivo. We demonstrate that cell-membrane-mimicking phosphatidylcholine (PC)-terminated monolayers improve the performance of E-AB sensors, reducing the baseline drift from around 70% to just a few percent after several hours in flowing whole blood in vitro. With this improvement comes the ability to deploy E-AB sensors directly in situ in the veins of live animals, achieving micromolar precision over many hours without the use of physical barriers or active drift-correction algorithms.

Craving elicited by drug-associated cues intensifies across protracted drug abstinence – a phenomenon termed “incubation of craving” – and drug-craving in human addicts correlates with frontal cortical hyperactivity. Herein, we employed a rat model of cue-elicited cocaine-craving to test the hypothesis that the time-dependent incubation of cue-elicited cocaine-craving is associated with adaptations in dopamine and glutamate neurotransmission within the ventromedial prefrontal cortex (vmPFC). Rats were trained to self-administer intravenous cocaine (6h/day × 10 days) and underwent in vivo microdialysis procedures during 2h-tests for cue-elicited cocaine-craving at either 3 or 30 days withdrawal. Controls rats were trained to either self-administer sucrose pellets or received no primary reinforcer. Cocaine-seeking rats exhibited a withdrawal-dependent increase and decrease, respectively, in cue-elicited glutamate and dopamine release. These patterns of neurochemical change were not observed in either control condition. Thus, cue-hypersensitivity of vmPFC glutamate terminals is a biochemical correlate of incubated cocaine-craving that may stem from dopamine dysregulation in this region.

In individuals with a history of drug-taking, the capacity of drug-associated cues to elicit indices of drug-craving intensifies or incubates with the passage of time during drug abstinence. This incubation of cocaine-craving, as well as difficulties with learning to suppress drug-seeking behavior during protracted withdrawal, are associated with a time-dependent deregulation of ventromedial prefrontal cortex (vmPFC) function. As the molecular bases for cocaine-related vmPFC deregulation remain elusive, the present study assayed the consequences of extended access to intravenous cocaine (6 h/d; 0.25 mg/infusion for 10 d) on the activational state of protein kinase C epsilon (PKCε), an enzyme highly implicated in drug-induced neuroplasticity. The opportunity to engage in cocaine-seeking during cocaine abstinence time-dependently altered PKCε phosphorylation within vmPFC, with reduced and increased p-PKCε expression observed in early (3 days) and protracted (30 days) withdrawal, respectively. This effect was more robust within the ventromedial versus dorsomedial PFC, was not observed in comparable cocaine-experienced rats not tested for drug-seeking behavior and was distinct from the rise in phosphorylated extracellular signal-regulated kinase (ERK) observed in cocaine-seeking rats. Further, the impact of inhibiting PKCε translocation within the vmPFC using TAT infusion proteins upon cue-elicited responding was determined and inhibition coinciding with the period of testing attenuated cocaine-seeking behavior, with an effect also apparent the next day. In contrast, inhibitor pretreatment prior to testing during early withdrawal was without effect. Thus, a history of excessive cocaine-taking influences the cue-reactivity of important intracellular signaling molecules within the vmPFC, with PKCε playing a critical role in the manifestation of cue-elicited cocaine-seeking during protracted drug withdrawal.

Phosphatidylinositide 3-kinases (PI3Ks) are intracellular signal transducer enzymes that recruit protein kinase B (aka Akt) to the cell membrane, the subsequent activation of which regulates many cellular functions. PI3K/Akt activity is up-regulated within mesocorticolimbic structures in animal models of alcoholism, but less is known regarding PI3K/Akt activity in animal models of cocaine addiction. Given that prefrontal cortex (PFC) is grossly dysregulated in addiction, we studied how cocaine affects protein indices of PFC PI3K/Akt activity in rat and mouse models and examined the relevance of PI3K activity for cocaine-related learning. Immunoblotting of mouse medial PFC at 3 weeks withdrawal from a cocaine-sensitization regimen (seven injections of 30 mg/kg, intraperitoneal [IP]) revealed increased kinase activity, as did immunoblotting of tissue from the ventral PFC of rats with a history of long-access intravenous cocaine self-administration (0.25 mg/0.1 mL infusion; 10 days of 6 h/d cocaine access). Interestingly, increased Akt phosphorylation was observed in rat ventromedial PFC at both 3- and 30-day withdrawal only in animals re-exposed to cocaine-associated cues. A conditioned place-preference paradigm in mice and a cue-elicited drug-seeking test in rats were conducted to determine the functional relevance for elevated PI3K activity for addiction-related behavior. In both cases, an intra-PFC infusion of the PI3K inhibitor wortmannin (50μM) reduced drug-seeking behavior. Taken together, this cross-species, interdisciplinary, study provides convincing evidence that cocaine history produces an enduring increase in PI3K/Akt-dependent signaling within the more ventral aspect of the PFC that is relevant to behavioral reactivity to drug-associated cues/contexts. As such, PI3K inhibitors may well serve as an effective strategy for reducing drug cue reactivity and craving in cocaine addiction.

Previous studies have shown that female rats exhibit different patterns of drug seeking during multiple phases of cocaine addiction when compared with males. However, the underlying mechanisms for these sex differences remain largely unknown. Here, we used a cocaine self-administration/reinstatement model to examine neuronal activation, as determined by Fos expression, following cue-induced reinstatement of cocaine seeking in male and female rats. Fos expression revealed both similarities between sexes in some brain regions, as well as selective sexually dimorphic patterns. As compared to no cue control subjects, conditioned cues induced higher Fos expression in the Cg1 region of the anterior cingulate cortex, but lower expression in the nucleus accumbens in both males and females. Females exhibited higher Fos expression than males in multiple brain regions, including the agranular insular cortex, dorsal medial caudate-putamen, nucleus accumbens shell, ventral tegmental area, dorsal subiculum, and ventral CA1 and CA3 regions of the hippocampus. Notably, only Fos expression in the prelimbic cortex, nucleus accumbens shell, basolateral amygdala, and ventral subiculum correlated positively with lever responding in response to conditioned cues across males and females. These findings indicate that while sexually dimorphic Fos activation does occur, the relationship between cue-induced cocaine seeking and neuronal activation may be similar for males and females in key brain regions of the relapse circuit.