Putting forward a model for the role of the cerebellum in cocaine-induced pavlovian memory.

Substance Use Disorder (SUD) involves emotional, cognitive, and motivational dysfunction. Long-lasting molecular and structural changes in brain regions functionally and anatomically linked to the cerebellum, such as the prefrontal cortex, amygdala, hippocampus, basal ganglia, and ventral tegmental area, are characteristic of SUD. Direct and indirect reciprocal connectivity between the cerebellum and these brain regions can explain cerebellar roles in Pavlovian and reinforcement learning, fear memory, and executive functions. It is increasingly clear that the cerebellum modulates brain functions altered in SUD and other neuropsychiatric disorders that exhibit comorbidity with SUD. In the present manuscript, we review and discuss this evidence and present new research exploring the role of the cerebellum in cocaine-induced conditioned memory using chemogenetic tools (designer receptor exclusively activated by designer drug, DREADDs). Our preliminary data showed that inactivation of a region that includes the interposed and lateral deep cerebellar nuclei reduces the facilitating effect of a posterior vermis lesion on cocaine-induced preference conditioning. These findings support our previous research and suggest that posterior vermis damage may increase drug impact on the addiction circuitry by regulating activity in the DCN. However, they raise further questions that will also be discussed.

Neural correlates of cocaine-induced conditioned place preference in the posterior cerebellar cortex.

Introduction: Addictive drugs are potent neuropharmacological agents capable of inducing long-lasting changes in learning and memory neurocircuitry. With repeated use, contexts and cues associated with consumption can acquire motivational and reinforcing properties of abused drugs, triggering drug craving and relapse. Neuroplasticity underlying drug-induced memories takes place in prefrontal-limbic-striatal networks. Recent evidence suggests that the cerebellum is also involved in the circuitry responsible for drug-induced conditioning. In rodents, preference for cocaine-associated olfactory cues has been shown to correlate with increased activity at the apical part of the granular cell layer in the posterior vermis (lobules VIII and IX). It is important to determine if the cerebellum's role in drug conditioning is a general phenomenon or is limited to a particular sensory modality. Methods: The present study evaluated the role of the posterior cerebellum (lobules VIII and IX), together with the medial prefrontal cortex (mPFC), ventral tegmental area (VTA), and nucleus accumbens (NAc) using a cocaine-induced conditioned place preference procedure with tactile cues. Cocaine CPP was tested using ascending (3, 6, 12, and 24 mg/kg) doses of cocaine in mice. Results: Compared to control groups (Unpaired and Saline animals), Paired mice were able to show a preference for the cues associated with cocaine. Increased activation (cFos expression) of the posterior cerebellum was found in cocaine CPP groups and showed a positive correlation with CPP levels. Such increases in cFos activity in the posterior cerebellum significantly correlated with cFos expression in the mPFC. Discussion: Our data suggest that the dorsal region of the cerebellum could be an important part of the network that mediates cocaine-conditioned behavior.

Role of Perineuronal nets in the cerebellar cortex in cocaine-induced conditioned preference, extinction, and reinstatement.

Perineuronal nets (PNNs) are cartilage-like structures of extracellular matrix molecules that enwrap in a net-like manner the cell-body and proximal dendrites of special subsets of neurons. PNNs stabilize their incoming connections and restrict plasticity. Consequently, they have been proposed as a candidate mechanism for drug-induced learning and memory. In the cerebellum, PNNs surround Golgi inhibitory interneurons and both inhibitory and excitatory neurons in the deep cerebellar nuclei (DCN). Previous studies from the lab showed that cocaine-induced conditioned memory increased PNN expression in the granule cell layer of the posterior vermis. The present research aimed to investigate the role of cerebellar PNNs in cocaine-induced conditioned preference. For this purpose, we use the enzyme chondroitinase ABC (ChABC) to digest PNNs at different time points of the learning process to ascertain whether their removal can affect drug-induced memory. Our results show that PNN digestion using ChABC in the posterior vermis (Lobule VIII) did not affect the acquisition of cocaine-induced conditioned preference. However, the removal of PNNs in Lobule VIII -but not in the DCN- disrupted short-term memory of conditioned preference. Moreover, although PNN digestion facilitated the formation of extinction, reinstatement of cocaine-induced conditioned preference was encouraged under PNN digestion. The present findings suggests that PNNs around Golgi interneurons are needed to maintain cocaine-induced Pavlovian memory but also to stabilize extinction memory. Conversely, PNN degradation within the DCN did not affect stability of cocaine-induced memories. Therefore, degradation of PNNs in the vermis might be used as a promising tool to manipulate drug-induced memory.

When the front fails, the rear wins. Cerebellar correlates of prefrontal dysfunction in cocaine-induced memory in male rats.

Reciprocal pathways connecting the cerebellum to the prefrontal cortex provide a biological and functional substrate to modulate cognitive functions. Dysfunction of both medial prefrontal cortex (mPFC) and cerebellum underlie the phenotypes of several neuropsychiatric disorders that exhibit comorbidity with substance use disorder (SUD). In people with SUD, cue-action-reward associations appears to be particularly strong and salient, acting as powerful motivational triggers for craving and relapse. Studies of cue reactivity in human with SUD have shown cerebellar activations when drug-related cues are presented. Our preclinical research showed that cocaine-induced conditioned preference increases neural activity and upregulates perineuronal nets (PNNs) around Golgi interneurons in the posterior cerebellar cortex. In the present investigation, we aimed at evaluating cerebellar signatures of conditioned preference for cocaine when drug learning is established under mPFC impairment. We used lidocaine to temporarily inactivate in male rats either the Prelimbic (PL) or the Infralimbic (IL) cortices during cocaine-induced conditioning. The inactivation of the IL, but not the PL, encouraged the acquisition of preference for cocaine-related cues, increased posterior cerebellar cortex activity, and upregulated the expression of PNNs around Golgi interneurons. Moreover, IL impairment not only increased vGluT2- and vGAT-related activity around Golgi cells but also regulated PNNs differently on subpopulations of Golgi cells, increasing the number of neurogranin+ PNN-expressing Golgi cells. Our findings suggest that IL dysfunction may facilitate the acquisition of cocaine-induced memory and cerebellar drug-related learning hallmarks. Overall, IL perturbation during cocaine-induced Pavlovian learning increased cerebellar activity and drug effects. Importantly, cerebellum involvement requires a contingent experience with the drug, and it is not the effect of a mere inactivation of IL cortex.

Time-dependent regulation of perineuronal nets in the cerebellar cortex during abstinence of cocaine-self administration.

RATIONALE: The probability of structural remodeling in brain circuits may be modulated by molecules of perineuronal nets (PNNs) that restrict neuronal plasticity to stabilize circuits. Animal research demonstrates that addictive drugs can remodel PNNs in different brain regions, including the cerebellum. OBJECTIVE: This study aimed to investigate the effects of short versus extended access to cocaine self-administration on PNN expression around Golgi interneurons in the cerebellar cortex after different periods of abstinence. METHODS: After 1 week of training (2 h/day), Sprague-Dawley rats self-administered cocaine daily for 20 days under short (ShA) or extended (LgA) access. PNN expression in the cerebellum was assessed after 1 day, 7 days, and 28 days of forced abstinence. PNNs were immunolabeled using Wisteria floribunda agglutinin (WFA) and captured by confocal microscopy. RESULTS: WFA intensity increased in PNN-bearing Golgi neurons over the abstinence period and a higher proportion of more intense PNNs were formed throughout the first month of abstinence. After the first 24 h of cocaine abstinence, however, we found a reduction in WFA intensity in the cerebellar cortex of rats with ShA to cocaine as compared to naïve animals. When comparing with naïve rats, LgA rats showed consistent PNN upregulation at 28 days of cocaine abstinence. CONCLUSIONS: Our results suggest that cocaine self-administration produces modifications in PNN that enhance conditions for synaptic plasticity in the cerebellar cortex. These modifications are revealed shortly after the cessation of drug intake but PNNs become more intense during protracted abstinence in the LgA group, pointing to the stabilization of drug-induced synaptic changes. These findings indicate that extended access to cocaine self-administration dynamically regulates conditions for plasticity in the cerebellum during abstinence.

From back to front: A functional model for the cerebellar modulation in the establishment of conditioned preferences for cocaine-related cues.

It is now increasingly clear that the cerebellum may modulate brain functions altered in drug addiction. We previously demonstrated that cocaine-induced conditioned preference increased activity at the dorsal posterior cerebellar vermis. Unexpectedly, a neurotoxic lesion at this region increased the probability of cocaine-induced conditioned preference acquisition. The present research aimed at providing an explanatory model for such as facilitative effect of the cerebellar lesion. First, we addressed a tracing study in which we found a direct projection from the lateral (dentate) nucleus to the ventral tegmental area (VTA) that also receives Purkinje axons from lobule VIII in the vermis. This pathway might control the activity and plasticity of the cortico-striatal circuitry. Then we evaluated cFos expression in different regions of the medial prefrontal cortex and striatum after a lesion in lobule VIII before conditioning. Additionally, perineuronal net (PNN) expression was assessed to explore whether the cerebellar lesion might affect synaptic stabilization mechanisms in the medial prefrontal cortex (mPFC). Damage in this region of the vermis induced general disinhibition of the mPFC and striatal subdivisions that receive dopaminergic projections, mainly from the VTA. Moreover, cerebellar impairment induced an upregulation of PNN expression in the mPFC. The major finding of this research was to provide an explanatory model for the function of the posterior cerebellar vermis on drug-related memory. In this model, damage of the posterior vermis would release striatum-cortical networks from the inhibitory tonic control exerted by the cerebellar cortex over VTA, thereby promoting drug effects.

The Cerebellum on Cocaine.

The traditional cerebellum's role has been linked to the high computational demands for sensorimotor control. However, several findings have pointed to its involvement in executive and emotional functions in the last decades. First in 2009 and then, in 2016, we raised why we should consider the cerebellum when thinking about drug addiction. A decade later, mounting evidence strongly suggests the cerebellar involvement in this disorder. Nevertheless, direct evidence is still partial and related mainly to drug-induced reward memory, but recent results about cerebellar functions may provide new insights into its role in addiction. The present review does not intend to be a compelling revision on available findings, as we did in the two previous reviews. This minireview focuses on specific findings of the cerebellum's role in drug-related reward memories and the way ahead for future research. The results discussed here provide grounds for involving the cerebellar cortex's apical region in regulating behavior driven by drug-cue associations. They also suggest that the cerebellar cortex dysfunction may facilitate drug-induced learning by increasing glutamatergic output from the deep cerebellar nucleus (DCN) to the ventral tegmental area (VTA) and neural activity in its projecting areas.

A Working Hypothesis for the Role of the Cerebellum in Impulsivity and Compulsivity.

Growing evidence associates cerebellar abnormalities with several neuropsychiatric disorders in which compulsive symptomatology and impulsivity are part of the disease pattern. Symptomatology of autism, addiction, obsessive-compulsive (OCD), and attention deficit/hyperactivity (ADHD) disorders transcends the sphere of motor dysfunction and essentially entails integrative processes under control of prefrontal-thalamic-cerebellar loops. Patients with brain lesions affecting the cortico-striatum thalamic circuitry and the cerebellum indeed exhibit compulsive symptoms. Specifically, lesions of the posterior cerebellar vermis cause affective dysregulation and deficits in executive function. These deficits may be due to impairment of one of the main functions of the cerebellum, implementation of forward internal models of the environment. Actions that are independent of internal models may not be guided by predictive relationships or a mental representation of the goal. In this review article, we explain how this deficit might affect executive functions. Additionally, regionalized cerebellar lesions have been demonstrated to impair other brain functions such as the emergence of habits and behavioral inhibition, which are also altered in compulsive disorders. Similar to the infralimbic cortex, clinical studies and research in animal models suggest that the cerebellum is not required for learning goal-directed behaviors, but it is critical for habit formation. Despite this accumulating data, the role of the cerebellum in compulsive symptomatology and impulsivity is still a matter of discussion. Overall, findings point to a modulatory function of the cerebellum in terminating or initiating actions through regulation of the prefrontal cortices. Specifically, the cerebellum may be crucial for restraining ongoing actions when environmental conditions change by adjusting prefrontal activity in response to the new external and internal stimuli, thereby promoting flexible behavioral control. We elaborate on this explanatory framework and propose a working hypothesis for the involvement of the cerebellum in compulsive and impulsive endophenotypes.

The role of the cerebellum in drug-cue associative memory: functional interactions with the medial prefrontal cortex.

Drug-induced Pavlovian memories are thought to be crucial for drug addiction because they guide behaviour towards environments with drug availability. Drug-related memory depends on persistent changes in dopamine-glutamate interactions in the medial prefrontal cortex (mPFC), basolateral amygdala, nucleus accumbens core and hippocampus. Recent evidence from our laboratory indicated that the cerebellum is also a relevant node for drug-cue associations. In the present study, we tested the role that specific regions of the cerebellum and mPFC play in the acquisition of cocaine-induced preference conditioning. Quinolinic acid was used to manage a permanent deactivation of lobule VIII in the vermis prior to conditioning. Additionally, lidocaine was infused into the prelimbic and infralimbic (IL) cortices for reversible deactivation before every training session. The present findings show, for the first time, that the cerebellum and mPFC might act together in order to acquire drug-cue Pavlovian associations. Either a dorsal lesion in lobule VIII or an IL deactivation encouraged cocaine-induced preference conditioning. Moreover, simultaneous IL-cerebellar deactivation prevented the effect of either of the separate deactivations. Therefore, similar to the IL cortex, neural activity in the cerebellum may be crucial for ensuring inhibitory control of the expression of cocaine-related memories.

Cocaine-Induced Preference Conditioning: a Machine Vision Perspective.

Existing work on drug-induced synaptic changes has shown that the expression of perineuronal nets (PNNs) at the cerebellar cortex can be regulated by cocaine-related memory. However, these studies on animals have mostly relied on limited manually-driven procedures, and lack some more rigorous statistical approaches and more automated techniques. In this work, established methods from computer vision and machine learning are considered to build stronger evidence of those previous findings. To that end, an image descriptor is designed to characterize PNNs images; unsupervised learning (clustering) is used to automatically find distinctive patterns of PNNs; and supervised learning (classification) is adopted for predicting the experiment group of the mice from their PNN images. Experts in neurobiology, who were not aware of the underlying computational procedures, were asked to describe the patterns emerging from the automatically found clusters, and their descriptions were found to align surprisingly well with the two types of PNN images revealed from previous studies, namely strong and weak PNNs. Furthermore, when the set of PNN images corresponding to every mice in the saline (control) group and the conditioned (experimental) group were characterized using a bag-of-words representation, and subject to supervised learning (saline vs conditioned mice), the high classification results suggest the ability of the proposed representation and procedures in recognizing these groups. Therefore, despite the limited size of the dataset (1,032 PNN images of 6 saline and 6 conditioned mice), the results support existing evidence on the drug-related brain plasticity, while providing higher objectivity.

Cerebellar perineuronal nets in cocaine-induced pavlovian memory: Site matters.

One of the key mechanisms for the stabilization of synaptic changes near the end of critical periods for experience-dependent plasticity is the formation of specific lattice extracellular matrix structures called perineuronal nets (PNNs). The formation of drug memories depends on local circuits in the cerebellum, but it is unclear to what extent it may also relate to changes in their PNN. Here, we investigated changes in the PNNs of the cerebellum following cocaine-induced preference conditioning. The formation of cocaine-related preference memories increased expression of PNN-related proteins surrounding Golgi inhibitory interneurons as well as that of cFos in granule cells at the apex of the cerebellar cortex. In contrast, the expression of PNNs surrounding projection neurons in the medial deep cerebellar nucleus (DCN) was reduced in all cocaine-treated groups, independently of whether animals expressed a preference for cocaine-related cues. Discriminant function analysis confirmed that stronger PNNs in Golgi neurons and higher cFos levels in granule cells of the apex might be considered as the cerebellar hallmarks of cocaine-induced preference conditioning. Blocking the output of cerebellar granule cells in α6Cre-Cacna1a mutant mice prevented re-acquisition, but not acquisition, of cocaine-induced preference conditioning. Interestingly, this impairment in consolidation was selectively accompanied by a reduction in the expression of PNN proteins around Golgi cells. Our data suggest that PNNs surrounding Golgi interneurons play a role in consolidating drug-related memories.