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Studies using rodent models have shown that relapse to drug or food seeking increases progressively during abstinence, a phenomenon termed ‘incubation of craving’. Mechanistic studies of incubation of craving have focused on specific neurobiological targets within pre- selected brain areas. Recent methodological advances in whole-brain immunohistochemistry, clearing, and imaging now enable unbiased brain-wide cellular resolution mapping of regions and circuits engaged during learned behaviors. However, these whole brain imaging approaches were developed for mouse brains while incubation of drug craving has primarily been studied in rats and incubation of food craving has not been demonstrated in mice. Here, we established a mouse model of incubation of palatable food craving and examined food reward seeking after 1, 15, and 60 abstinence days. We then used the neuronal activity marker Fos with intact brain mapping procedures to identify corresponding patterns of brain-wide activation. Relapse to food seeking was significantly higher after 60 abstinence days than after 1 or 15 days. Using unbiased ClearMap analysis, we identified increased activation of multiple brain regions, particularly corticostriatal structures, following 60, but not 15 abstinence days. We used orthogonal SMART2 analysis to confirm these findings within corticostriatal and thalamocortical subvolumes and applied expert-guided registration to investigate subdivision and layer-specific activation patterns. Overall, we (1) identified novel brain-wide activity patterns during incubation of food seeking using complementary analytical approaches, and (2) provide a single-cell resolution whole-brain atlas that can be used to identify functional networks and global architecture underlying incubation of food craving.Significance StatementRelapse to reward seeking progressively increases during abstinence, a phenomenon termed incubation of craving. Mechanistic studies of incubation can lead to novel relapse treatments. However, previous studies have primarily used rat models and targeted region-by-region analyses and a brain-wide functional atlas of incubation of reward seeking is lacking. We established a behavioral procedure for incubation of palatable food seeking in mice and applied whole-brain activity mapping with Fos as a neuronal activity marker to identify the functional connectome of this incubation. Like rats, mice showed incubation of food seeking during abstinence. Using two complementary activity mapping approaches, we identified a brain-wide pattern of increased neural activation that mirrored incubation of food seeking after 60, but not 15, days of abstinence.

Persistent susceptibility to cue-induced relapse is a cardinal feature of addiction. Discriminative stimuli (DSs) are one type of drug-associated cue that signal drug availability (DS+) or unavailability (DS−) and control drug seeking prior to relapse. We previously established a trial-based procedure in rats to isolate DSs from context, conditioned stimuli, and other drug-associated cues during cocaine self-administration and demonstrated DS-controlled cocaine seeking up to 300 abstinence days. The behavioral and neural mechanisms underlying trial-based DS-control of drug seeking have rarely been investigated. Here we show that following discrimination training in our trial-based procedure, the DS+ and DS− independently control the expression and suppression of cocaine seeking during abstinence. Using microinjections of GABA(A) + GABA(B) receptor agonists (muscimol + baclofen) in medial prefrontal cortex, we report that infralimbic, but not prelimbic, subregion of medial prefrontal cortex is critical to persistent DS-controlled relapse to cocaine seeking after prolonged abstinence, but not DS-guided discriminated cocaine seeking or DS-controlled cocaine self-admininstration. Finally, using ex vivo whole-cell recordings from pyramidal neurons in the medial prefrontal cortex, we demonstrate that the disruption of DS-controlled cocaine seeking following infralimbic cortex microinjections of muscimol+baclofen is likely a result of suppression of synaptic transmission in the region via a presynaptic mechanism of action.

α-Synuclein is an abundant protein at the neuronal synapse that has been implicated in Parkinson's disease for over 25 years and characterizes the hallmark pathology of a group of neurodegenerative diseases now known as the synucleinopathies. Physiologically, α-synuclein exists in an equilibrium between a synaptic vesicle membrane-bound α-helical multimer and a cytosolic largely unstructured monomer. Through its membrane-bound state, α-synuclein functions in neurotransmitter release by modulating several steps in the synaptic vesicle cycle, including synaptic vesicle clustering and docking, SNARE complex assembly, and homeostasis of synaptic vesicle pools. These functions have been ascribed to α-synuclein's interactions with the synaptic vesicle SNARE protein VAMP2/synaptobrevin-2, the synaptic vesicle-attached synapsins, and the synaptic vesicle membrane itself. How α-synuclein affects these processes, and whether disease is due to loss-of-function or gain-of-toxic-function of α-synuclein remains unclear. In this review, we provide an in-depth summary of the existing literature, discuss possible reasons for the discrepancies in the field, and propose a working model that reconciles the findings in the literature.

α-synuclein, β-synuclein, and γ-synuclein are abundantly expressed proteins in the vertebrate nervous system. α-synuclein functions in neurotransmitter release by binding to and clustering synaptic vesicles and chaperoning SNARE-complex assembly. Pathologically, aggregates originating from soluble pools of α-synuclein are deposited into Lewy bodies in Parkinson’s disease and related synucleinopathies. The functions of β-synuclein and γ-synuclein in presynaptic terminals remain poorly studied. Using in vitro liposome binding studies, circular dichroism spectroscopy, immunoprecipitation, and fluorescence resonance energy transfer (FRET) experiments on isolated synaptic vesicles in combination with subcellular fractionation of brains from synuclein mouse models, we show that β-synuclein and γ-synuclein have a reduced affinity toward synaptic vesicles compared with α-synuclein, and that heteromerization of β-synuclein or γ-synuclein with α-synuclein results in reduced synaptic vesicle binding of α-synuclein in a concentration-dependent manner. Our data suggest that β-synuclein and γ-synuclein are modulators of synaptic vesicle binding of α-synuclein and thereby reduce α-synuclein’s physiological activity at the neuronal synapse.

SUMMARYα-Synuclein (αSyn), β-synuclein (βSyn), and γ-synuclein (γSyn) are abundantly expressed in the vertebrate nervous system. αSyn functions in neurotransmitter release via binding to and clustering synaptic vesicles and chaperoning of SNARE-complex assembly. The functions of βSyn and γSyn are unknown. Functional redundancy of the three synucleins and mutual compensation when one synuclein is deleted have been proposed, but with conflicting evidence. Here, we demonstrate that βSyn and γSyn have a reduced affinity towards membranes compared to αSyn, and that direct interaction of βSyn or γSyn with αSyn results in reduced membrane binding of αSyn. Our data suggest that all three synucleins affect synapse function, but only αSyn mediates the downstream function of vesicle clustering and SNARE-complex assembly, while βSyn and γSyn modulate the activity of αSyn through regulating its binding to synaptic vesicles.

Recent studies suggest that the ventral medial prefrontal cortex (vmPFC) encodes both operant drug self-administration and extinction memories. Here, we examined whether these opposing memories are encoded by distinct neuronal ensembles within the vmPFC with different outputs to the nucleus accumbens (NAc) in male and female rats. Using cocaine self-administration (3 h/d for 14 d) and extinction procedures, we demonstrated that vmPFC was similarly activated (indexed by Fos) during cocaine-seeking tests after 0 (no-extinction) or 7 extinction sessions. Selective Daun02 lesioning of the self-administration ensemble (no-extinction) decreased cocaine seeking, whereas Daun02 lesioning of the extinction ensemble increased cocaine seeking. Retrograde tracing with fluorescent cholera toxin subunit B injected into NAc combined with Fos colabeling in vmPFC indicated that vmPFC self-administration ensembles project to NAc core while extinction ensembles project to NAc shell. Functional disconnection experiments (Daun02 lesioning of vmPFC and acute dopamine D1-receptor blockade with SCH39166 in NAc core or shell) confirm that vmPFC ensembles interact with NAc core versus shell to play dissociable roles in cocaine self-administration versus extinction, respectively. Our results demonstrate that neuronal ensembles mediating cocaine self-administration and extinction comingle in vmPFC but have distinct outputs to the NAc core and shell that promote or inhibit cocaine seeking. SIGNIFICANCE STATEMENT Neuronal ensembles within the vmPFC have recently been shown to play a role in self-administration and extinction of food seeking. Here, we used the Daun02 chemogenetic inactivation procedure, which allows selective inhibition of neuronal ensembles identified by the activity marker Fos, to demonstrate that different ensembles for cocaine self-administration and extinction memories coexist in the ventral mPFC and interact with distinct subregions of the nucleus accumbens.

In abstinent drug addicts, cues formerly associated with drug-taking experiences gain relapse-inducing potency (‘incubate’) over time. Animal models of incubation may help develop treatments to prevent relapse, but these models have ubiquitously focused on the role of conditioned stimuli (CSs) signaling drug delivery. Discriminative stimuli (DSs) are unique in that they exert stimulus-control over both drug taking and drug seeking behavior and are difficult to extinguish. For this reason, incubation of the excitatory effects of DSs that signal drug availability, not yet examined in preclinical studies, could be relevant to relapse prevention. We trained rats to self-administer cocaine (or palatable food) under DS control, then investigated DS-controlled incubation of craving, in the absence of drug-paired CSs. DS-controlled cocaine (but not palatable food) seeking incubated over 60 days of abstinence and persisted up to 300 days. Understanding the neural mechanisms of this DS-controlled incubation holds promise for drug relapse treatments., eLife digest More than 85% of people who give up an addictive drug begin using it again within a year. Relapse rates have changed little over the past five decades. Situations, places and objects associated with drug-taking can trigger relapse long after a person’s last exposure to a drug. We can study relapse by training animals to self-administer drugs such as cocaine. For example, rats can learn to press a lever to receive an infusion of a drug paired with a cue (a conditioned stimulus), such as a tone or a light. After training, the rats continue to press the lever to seek the drug, even if this behavior no longer delivers it. In addition, their lever pressing in response to cues increases with time for several months after their last drug exposure. This phenomenon, known as incubation of drug craving, mirrors the increase in cravings reported by abstinent drug users. In drug users, cues such as the crack pipe or syringe used to take the drug can later contribute to drug relapse during abstinence. Most studies modeling this phenomenon have focused on how the rats respond to a conditioned stimulus that signaled the delivery of a drug during training. However, a second type of signal, known as the discriminative stimulus, can also influence relapse. Discriminative stimuli are sets of cues that signal whether drugs are about to become available or not; for example, the presence of people selling drugs on a street corner as opposed to the presence of police. Madangopal et al. now show that discriminative stimuli – in the absence of conditioned stimuli – can also control the incubation of drug craving. Rats learned to press a lever in response to a light signaling the availability of cocaine (the positive discriminative stimulus), and to avoid responding to a different light indicating that cocaine was unavailable (the negative discriminative stimulus). When tested during abstinence, the rats only increased their lever pressing to the first light over time, i.e., they showed an incubation of drug craving controlled by the positive discriminative stimulus. Lever pressing peaked after 60 days of abstinence and persisted for up to 300 days (almost half the rats' lifespan). By contrast, the same discriminative stimuli did not trigger increased lever pressing when used to signal the availability of a palatable food. Discriminative stimuli are thus powerful and persistent triggers of craving for addictive drugs. They signal the availability of a drug prior to both drug-taking and relapse, making them a critical target for intervention strategies. Understanding the mechanisms by which discriminative stimuli promote drug craving could lead to new treatments to prevent relapse.

In abstinent drug addicts, cues formerly associated with drug-taking experiences gain relapse-inducing potency (‘incubate’) over time. Animal models of incubation may help in developing treatments for relapse prevention. However, these models have primarily focused on the role of conditioned stimuli (CSs) signaling drug delivery and not on discriminative stimuli (DSs), which signal drug availability and are also known to play a major role in drug relapse. We recently showed that DS-controlled cocaine seeking in rats also incubates during abstinence and persists up to 300 days. We used a trial-based procedure to train male and female rats to discriminate between two light cues: one light cue (DS+) signaled the availability of cocaine reward and the second light cue (DS-) signaled the absence of reward. Rats learned to press a central retractable lever during trials in which the DS+ cue was presented and to suppress responding when the DS- cue was presented. Here, we provide a detailed protocol for the behavioral procedure used in our study. The trial-based design of this behavior lends itself well to time-locked in vivo recording and manipulation approaches that can be used to identify neurobiological mechanisms underlying the contributions of DSs to drug relapse.

In abstinent drug addicts, cues formerly associated with drug-taking experiences gain relapse-inducing potency ('incubate') over time. Animal models of incubation may help develop treatments to prevent relapse, but these models have ubiquitously focused on the role of conditioned stimuli (CSs) signaling drug delivery. From a translational perspective this is problematic because people encounter these stimuli only during or after relapse. For this reason, incubation in response to discriminative stimuli (DSs) that signal drug availability before relapse, not yet examined in preclinical studies, could be more relevant to relapse prevention. We trained rats to self-administer cocaine (or palatable food) under DS control, then investigated DS-controlled incubation of craving, in the absence of drug-paired CSs. DS-controlled cocaine (but not palatable food) seeking incubated over 60 days of abstinence and persisted up to 300 days. Understanding the neural mechanisms of this DS-controlled incubation holds significant promise for drug relapse treatments.