{'fraction_non_alphanumeric': 0.04116763584549621, 'fraction_numerical': 0.03146301074440636, 'mean_word_length': 4.955963302752294, 'pattern_counts': {'":': 0, '<': 3, '<?xml version=': 0, '>': 1, 'https://': 0, 'lorem ipsum': 0, 'www.': 1, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Time-dependent regulation of perineuronal nets in the cerebellar cortex during abstinence of cocaine-self administration

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. 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. 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. 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. 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.


Introduction
The probability of structural remodeling in brain circuits may be modulated by the activity of regulatory molecules that restrict neuronal plasticity to stabilize circuits (Foscarin et al. 2011). This regulatory activity occurs in lattice cartilage-like structures called perineuronal net (PNN) consisting of molecules of extracellular matrix (ECM) that wrap several neuronal populations, the majority of them fast-spiking GABAergic interneurons (Grimpe and Silver 2002;Carulli et al. 2006;Soleman et al. 2013;Sorg et al. 2016;Miyata and Kitagawa 2017;Fawcett et al. 2019).
Several PNN components can interact with membrane receptors and ion channels, and change electrophysiological properties and plasticity events of PNN-bearing neurons (Fawcett et al. 2019). In particular, PNNs appear to funnel or restrict the number of possible future plastic events and to represent a form of cellular memory (Dityatev and Schachner 2003). Consequently, PNN digestion with chondroitinase ABC (ChABC), an enzyme that targets the chondroitin sulfate proteoglycans (CSPGs), causes the reversion to an immature brain stage, thus increasing plasticity conditions (Pizzorusso et al. 2002;Lensjø et al. 2017). Both inhibitory and excitatory balance (Lensjø et al. 2017), and AMPA receptor mobility (Frischknecht et al. 2009;Favuzzi et al. 2017) are all affected after PNN removal. Therefore, PNNs participate in maintaining the synaptic architecture critical to neural plasticity, which is believed to mediate learning and memory, and thereby, it could underlie drug addiction (Wright and Harding 2009;Fawcett et al. 2019).
Findings from animal and human research evidence the ability of addictive drugs to remodel PNNs dynamically. Drug-related plasticity and metaplasticity involve PNN regulation in different brain regions (Mash et al. 2007; Van den Oever et al. 2010;Xue et al. 2014;Chen et al. 2015;Slaker et al. 2015Slaker et al. , 2016Vazquez-Sanroman et al. 2017;Blacktop et al. 2017;Slaker et al. 2018;Blacktop and Sorg 2019;Gil-Miravet et al. 2019), including the cerebellum (Vazquez-Sanroman et al. 2015a, b;Carbo-Gas et al. 2017). Druginduced remodeling of PNNs depends on several factors such as the brain region, the length of the withdrawal period, the time course, and the behavioral process investigated. Indeed, whereas chronic experience with cocaine and alcohol increases PNN expression and, thus, Wisteria floribunda agglutinin (WFA) staining in the prelimbic cortex (Slaker et al. 2018), cerebellum (Vazquez-Sanroman et al. 2015b;Carbo-Gas et al. 2017), and insula (Chen et al. 2015), nicotine selfadministration reduces WFA staining in the VTA and orbitofrontal cortex ). Removal of PNNs in these brain regions has revealed the functional role of this specialized ECM in the acquisition, reinstatement (Slaker et al. 2015;Blacktop et al. 2017), and extinction (Xue et al. 2014) of drug-related behaviors.
A neurotoxic lesion of the cerebellum's posterior vermis has shown to facilitate cocaine-induced preference conditioning and to increase neural activity and PNN expression in GABAergic parvalbumin + interneurons of the medial prefrontal cortex (Gil-Miravet et al. 2019). In the cerebellar cortex, cocaine-induced conditioned preference has been associated with more intense PNNs around Golgi interneurons (Carbo-Gas et al. 2017;Traver et al. 2018). However, cocaine-dependent regulation of PNNs surrounding output neurons in the deep cerebellar nuclei (DCN) was not linked to drug memory (Carbo-Gas et al. 2017) but to neuroplasticity events that occur during abstinence periods. Cocaine-induced reinstatement increases PNN expression after short abstinence (Vazquez-Sanroman et al. 2015a) but decreases its expression after protracted abstinence (Vazquez-Sanroman et al. 2015b) in the DCN. Therefore, through PNN regulation, addictive drugs may affect synaptic plasticity and the conditions for plasticity, creating restrictive or permissive metaplasticity (Slaker et al. 2015;Vazquez-Sanroman et al. 2015a;Neuhofer and Kalivas 2018).
In the present research, we investigated the effects of short versus extended access to cocaine self-administration, a model of development of addiction (Ahmed and Koob 1998), on PNN expression in the cerebellum. PNN expression was evaluated after 1 day, 7 days, or 28 days of abstinence from cocaine self-administration. We aimed at exploring whether long-term cocaine exposure can remodel PNNs around Golgi inhibitory interneurons and whether PNN regulation persists after protracted abstinence.

Subjects and housing conditions
Our materials and methods are the same described in Nicolas et al. (2017) for all behavioral experiments. Adult male Sprague-Dawley rats (8-10 weeks) (N = 40) (Janvier Labs, France), experimentally naïve at the beginning of the study, were housed in a temperature and humidity-controlled environment and maintained on 12-h light/dark cycle (light on at 7 AM). All experiments were conducted following the European Union directives (2010/63/EU) for the care of laboratory animals and approved by the local ethics committees (COMETHEA). Upon arrival, rats were housed two per cage for about 1 week before intra-jugular vein catheterization surgery. After surgery, rats were housed individually for the entire period of self-administration and 3 per cage during the abstinence period. Naïve-control rats had the same housing conditions. We chose these housing conditions because social isolation is a well-known stressors in rodents that influences addiction-related processes and social housing appears a better control for the effects of environmental enrichment (see Solinas et al. 2010 for discussion). Both our group and other groups have used social housing during abstinence and found long-lasting cocaine seeking and incubation of cocaine craving (Chauvet et al. 2009;Chauvet et al. 2012;Thiel et al. 2009aThiel et al. , b, 2011. was fixed to the guide cannula with thermoretractable tubing (Conrad, France) and encased in dental cement anchored with a 2.5-cm 2 durable mesh used as a subcutaneous anchor. The tubing was inserted subcutaneously from the animals back to the right external jugular vein. After surgery, all animals could recover for a minimum of 5 days before given access to cocaine during which catheters were flushed daily with 0.15 ml of a filtered heparinized saline solution 0.6% (Sigma, St. Quentin Fallavier, France) and gentamicin 4% (Panpharma, Luitré, France).

Self-administration apparatus and procedure
The experimental timeline is schematized in Fig. 1. A week after catheter surgery, self-administration training began in operant chambers (Imetronic, Pessac, France) equipped with nose-pokes as operanda and controlled by Imetronic interfaces and software (Imetronic, Pessac, France, www. imetronic.com). Rats were allowed to self-administer cocaine according to a fixed ratio 1 (FR1) schedule of reinforcement. Thus, an active nose-poke response immediately resulted in an IV injection of cocaine for 5 s (0.3 mg/ injection), the blinking of house light for 5 s followed by a 5-s time-out period during which the chamber was dark, and further active nose-pokes had no programmed consequences. The first 7 sessions lasted 2 h for all rats. Starting from the 8th session, rats were pseudo-randomly divided into two groups after the criterion for the acquisition was reached (at least 15 self-infusions in 2 h) (Ahmed and Koob 1998): (i) short-access group (ShA), with access to cocaine self-administration for 1 h per day and (ii) longaccess group (LgA) with access to cocaine selfadministration for 6 h/day, for 20 sessions. These two paradigms of drug exposure are designed to mimic recreational versus addictive cocaine intake, respectively (Ahmed and Koob 1998). Then, ShA and LgA rats were left undisturbed in their home cages for 1, 7, or 28 days before perfusion. A group of naïve rats paired for age and housing conditions did not undergo any behavioral training and did not have exposure to cocaine and served as a control.

Perfusion and dissection protocol
Animals were deeply anesthetized with sodium pentobarbital (30 mg/kg) and perfused transcardially, first with 0.9% saline solution and 4% paraformaldehyde. After perfusion, the brain was immediately dissected and placed in a container with 4% paraformaldehyde for 24 h. After this time, tissue was cryoprotected in 30% sucrose solution until complete immersion. Brain tissue was rapidly frozen by immersion in liquid nitrogen, and coronal sections were performed at 40 μm with a cryostat (CM3050S, Leica, France). Five series of tissue sections were collected and stored at − 80°C in a cryoprotectant solution. Coronal sections of the cerebellum were selected for immunoanalysis.
PNNs were labeled and analyzed throughout the entire cerebellar cortex, including the vermis and hemispheres. Imaging and assessment of PNNs were blind to the treatment. In the cerebellar cortex, only Golgi inhibitory interneurons express a PNN (Corvetti and Rossi 2005;Carulli et al. 2006). To estimate changes in WFA intensity, we analyzed all PNNs that showed unquestionable integrity in their structure. A × 40 objective with a × 3.0 zoom was used for a final magnification of  Fig. 1 Experimental timeline. After 7 days of cocaine self-administration training (2 h/day), rats were randomly assigned to short access (ShA, 1 h/ day) and long access (LgA, 6 h/day). At the end of the 20 days of cocaine self-administration, rats underwent 1 day, 7 days, or 28 days of force abstinence. Naïve rats were used as controls × 5120. Brightness intensity of every PNN was estimated by randomly selecting 15 pixels in a single plane of the net pulled from a 40-μm z-stack and calculating their average intensity (Carbo-Gas et al. 2017). That is, we moved in the z-plane until we found the plane in which each PNN showed the highest integrity. This procedure enables to not underestimate intensity when the PNN appears in a different z-plane. Data were expressed as arbitrary units of intensity (AU of WFA intensity) with a maximum intensity of 255. Then, we selected 30 PNNs randomly per rat to calculate the proportion of strong (more intense) (171-255 AU), medium (86-170 AU) and faint (0-85 AU) PNNs. Pictures of fluorescent-labeled sections were acquired using a Leica SP8 confocal microscope. Image resolution was 1024 × 1024 and scan speed 600 Hz. Laser intensity, gain, and offset were maintained constant in each acquisition. All quantitative evaluations were made using the FIJI software (Schindelin et al. 2012).

Statistics
Results of cocaine self-administration were analyzed by twoway repeated-measures ANOVA with sessions as a withinsubject factor and cocaine access (ShA/LgA) as a betweensubject factor. One-way (group) and two-way (group x abstinence days) ANOVAs were used for the analysis of PNN intensity. Differences between means were analyzed through Tukey's HDS and Sidak's tests. We also classified PNNs in three categories, strong, medium, and weak, and compared the percentage of PNNs in each category using the χ 2 test. Data is presented as mean ± SD and 95% confidence intervals (CI). All the statistical analyses were performed using GraphPad Prism software 8 (San Diego, CA, USA).

Protracted abstinence increases PNN expression in the cerebellar cortex
To characterize the effects of short (n = 18) versus extended access (n = 18) to cocaine self-administration on cerebellar PNN expression during abstinence, we compared WFA intensity around Golgi interneurons 1, 7, or 28 days after the last cocaine self-administration session (Fig. 3). A two-way ANOVA (cocaine access × withdrawal time) yields significant differences for cocaine access (F(1, 10) = 13.71, P = 0.0041) and abstinence time (F(2, 20) = 11.46, P = 0.0005) but not for the interaction between these two factors (F(2, 20) = 0.3510, P = 0.7083). The lack of a significant interaction indicates that PNN upregulation through abstinence occurred in both cocaine groups. Nevertheless, as can be seen in Fig. 3a In addition, the distribution of strong and faint PNNs in the cerebellar cortex was different among groups (χ 2 (10) = 158.30, P < 0.0001). ShA rats expressed 81% of faint and 1.6% of more intense PNNs at 1 day after cocaine self-administration. After 28 days of abstinence, the percentage of faint PNNs decreased to 53% while strong PNNs increased to 22%. Moreover, 66% of PNNs were faint and 4% were strong 1 day after extended accesses to cocaine. Protracted abstinence reduced the proportion of faint PNNs (27%) but enhanced the proportion of strong (38%) (Fig. 3c).

Cocaine self-administration reduces PNN expression in the cerebellar cortex
To determine whether different history of cocaine selfadministration change PNN expression in the cerebellum, ShA and LgA rats were compared with naïve rats 1 day after the last cocaine self-administration session (Ctrl; n = 6, 83.44 ± 4.22, 95% CI [79.01,87.87]) (Fig. 4). Cocaine selfadministration under short-access conditions reduced WFA intensity in Golgi cells bearing a PNN (F(2,15) = 3.85, P = 0.0450). The reduction was clear in the ShA group so that the majority of subjects exhibited WFA intensity below control levels (P = 0.0470, DF: 15) (Fig. 4a). However, WFA intensity in the LgA group after 24 h did not differ from naïve rats (P = 0.8520, DF: 15) (Fig. 4b). We also found a different distribution of strong (> 170 AU of WFA) and faint (< 85 AU of WFA) PNNs among groups. While naïve rats expressed 63% of faint and 12% of strong PNNs, the ShA group exhibited 81% of faint PNNs but only 1.6% of strong  Fig. 3 Expression of PNNs in the cerebellar cortex at different time points of cocaine abstinence in ShA versus LgA rats. a Violin plots with individual scores of WFA intensity from PNNs throughout different periods (1, 7, and 28 days) of force abstinence in ShA and LgA rats. 1 day ShA (n = 6), 7 days ShA (n = 6), 28 days ShA (n = 6), 1 day LgA (n = 6), 7 days LgA (n = 6), and 28 days LgA (n = 6). Plots indicate data range, quartiles, and median. The Y-axis represents WFA intensity expressed in arbitrary units (AU). b Representative confocal images (× 120) of PNNs in each group. Protracted abstinence from cocaine increased PNN expression around Golgi interneurons in the cerebellar cortex. Scale bar 20 μm. c Proportion of Golgi neurons expressing a faint, medium, or strong PNNs in the cerebellar cortex ones (χ 2 (4) = 28.76, P < 0.0001). LgA rats showed 66% of faint and 4% of strong PNNs, and they were not different from naïve-control rats (Fig. 4c).

PNN expression did not return to control levels after protracted abstinence
To assess whether PNN expression returns to control levels after protracted abstinence, we compared ShA and LgA rats with naïve controls after 28 days of abstinence. Extended access to cocaine self-administration caused a significant increase in WFA intensity around Golgi cells that did not return to control levels after 28 days of abstinence (F(2, 15) = 8.220, P = 0.0039) (LgA: P = 0.0030; DF: 15). This effect was not evident in ShA rats (P = 0.3096, DF: 15) (Fig. 5). Finally, we did not find significant effects in the number of PNNs as a Kruskal-Wallis test showed (H(7) = 10.03, P = 0.1235; n = 3). We show mean ± SD and median with 95% confidence interval of median for all groups:

Discussion
In a previous investigation, we showed that the formation of cocaine-induced conditioned preference is associated with increases in PNN expression surrounding Golgi interneurons of the cerebellar cortex (Carbo-Gas et al. 2017). More intense PNNs correlated with a higher preference for cocaine-related cues (Carbo-Gas et al. 2017). In the present study, our results show a dynamic regulation of PNNs during abstinence in the cerebellar cortex. After the first 24 h of abstinence, we found a reduction in the expression of PNNs around these cerebellar GABAergic interneurons in animals with short access to cocaine. Over the course of prolonged abstinence, however, the expression of cerebellar PNNs increased, and a higher proportion of intense PNNs formed throughout the first month. The intensity of PNNs across time was not different in ShA rats, but it enhanced during abstinence after an escalated cocaine intake (LgA).
It has been hypothesized that PNNs play a role in regulating neural plasticity via three possible mechanisms: (1) altering the formation of new neuronal contacts (Corvetti and Rossi 2005;Barritt et al. 2006); (2) acting as a scaffold for molecules that can inhibit synaptic formation (Deepa et al. 2006);and (3) limiting receptor motility at synapses (Frischknecht et al. 2009). It is clear that reduction in the expression of PNNs can facilitate synaptic plasticity and enable synaptic remodeling (Pizzorusso et al. 2002;Lensjø et al. 2017;Carulli et al. 2020). Accordingly, degradation of PNNs around Golgi interneurons by the enzyme chondroitinase ABC increases structural plasticity in the cerebellar cortex (Corvetti and Rossi 2005). Our findings suggest that cocaine self-administration might enhance conditions for synaptic plasticity in this region that were revealed shortly after the cessation of drug intake (24 h) in ShA rats. The regulation of PNNs during this first 24 h after chronic cocaine is not the same in all brain regions. Nonetheless, any possible comparison among studies should be taken with caution because they involve distinct behavioral protocols for cocaine administration and diverse drug-induced memory processes. Density of high intensity PNNs increases in the right prelimbic, infralimbic, and ventral orbitofrontal cortex 24 h after cocaine self-administration (Roura-Martínez et al. 2020). Under the same conditions, the density of low-and medium-intensity PNNs was higher in the left hemisphere (Roura-Martínez et al. 2020). Moreover, PNN expression decreased in the infralimbic cortex, but it increased in the prelimbic cortex after 5 days of i.p. cocaine administration (Slaker et al. 2018). Endogenous enzymatic remodeling of PNNs relays on several families of proteases, including matrix metalloproteinases (MMPs) (Huntley 2012) and proteases that belong to the plasminogen-activating system such as the tissue plasminogen activator (tPA) (De Luca and Papa 2016). The activity of these enzymes is dynamically regulated during cocaine abstinence and reinstatement in several regions (Van den Oever et al. 2010;Smith et al. 2014;Vazquez-Sanroman et al. 2015b). Both MMP 2 and 9 have been detected in the cerebellum (Stamenkovic et al. 2017), although their role in druginduced plasticity and memory is unknown. In our previous research, we found that 24 h following a new i.p cocaine challenge administered after protracted abstinence (1 month), tPA activity enhanced and PNN expression was downregulated in the deep cerebellar nuclei (Vazquez-Sanroman et al. 2015b). No changes in tPA levels were detected following a shorter period of abstinence (1 week) (Vazquez-Sanroman et al. 2015a).
The present results also indicate that PNNs around Golgi interneurons increased their expression progressively during abstinence. More intense PNNs may stabilize synaptic modifications induced by the extended access to cocaine and enable their persistence during abstinence. Several of constituent molecules in a PNN such as brevican, neurocan, and tenascin-R are involved in the maintenance of short-and long-term plasticity (Brakebusch et al. 2002;see Fawcett et al. 2019 for a recent review). Strong PNNs might represent a common mechanism to maintain drug-related memories and, therefore, contribute to plasticity leading to relapse. In agreement with this hypothesis, the digestion of PNNs in the anterior hypothalamic area prevented reinstatement of cocaine self-administration, but it did not affect sucrose selfadministration (Blacktop et al. 2017). Moreover, degradation of PNNs into the amygdala during extinction training blocked reinstatement of cocaine-and morphine-induced conditioned place preference (Xue et al. 2014). In other words, strong PNNs might "stamp in" synaptic arrangements that represent drug-cue associations and contribute to long-lasting drug memories by preventing synaptic remodeling Lasek et al. 2018).
Golgi neurons are crucial elements of local circuits in the cerebellar cortex (see D'Angelo and De Zeeuw 2009; D'Angelo et al. 2013 for reviews). Their axons inhibit neighboring granule cells (GC) (Tabuchi et al. 2019) and play a crucial role in the synchronization of activity in GC clusters (Eccles et al. 1964). Golgi cell activity is controlled by glutamatergic inputs from GC and mossy fibers (Palay and Chan-Palay 1974), as well as by GABAergic and glycinergic inhibitory signals from other populations of cerebellar interneurons (Sotelo and Llinás 1972;Dumoulin et al. 2001). Golgi cells thus appear to be essential regulators of plasticity in the cerebellar cortex (D'Angelo et al. 1999;Armano et al. 2000). The present study is the first to involve Golgi cells bearing a PNN in plasticity linked to abstinence after cocaine self-administration. Our previous findings indicated that cocaine memory increases activity of GC and Golgi neurons, and generates strong and fully condense PNNs around these inhibitory interneurons after 48 h (Carbo-Gas et al. 2017). The expected consequence of increasing activity in GC is an enhanced inhibitory effect of molecular interneurons over Purkinje activity, since parallel fibers stimulate dendrites of molecular interneurons (Gao et al. 2016;Albergaria et al. 2018). Although in the present work, we did not evaluate activity changes in the cerebellar cortex, it is conceivable that the cerebellar cortex would present a similar pattern after protracted abstinence. Namely, stronger PNNs around Golgi neurons being associated with increased activity in GC. If this were the case, protracted cocaine abstinence would decrease Purkinje activity and facilitate the cerebellar output to other brain regions. It is now clear that the cerebellum may control the activity of dopaminergic and non-dopaminergic neurons within the VTA (Carta et al. 2019). Moreover, we have recently shown that a permanent perturbation of Purkinje activity by a neurotoxic lesion in the posterior cerebellar cortex facilitates the acquisition of cocaine-induced conditioned memory and increases neural activity in the DCN, VTA, ventral and dorsal striatum, and the medial prefrontal cortex (Gil-Miravet et al. 2019). Time-dependent increases in the ability of drug-related cues to trigger relapse during abstinence is known as the incubation effect (Grimm et al. 2001;Dong et al. 2017). The circuit involved in such effect includes regions of the VTA, the amygdala, the nucleus accumbens, and the medial prefrontal cortex (Dong et al. 2017). Overall, the most recent findings strongly point to a cerebellar modulation of these brain regions (Carta et al. 2019;Gil-Miravet et al. 2019). Further investigation will elucidate the particular role of cerebellar PNNs in the incubation of craving and relapse.

Concluding remarks
Even though several findings indicated the cerebellum's involvement in the functional alterations observed after prolonged drug use (Miquel et al. 2009;Moulton et al. 2014;Miquel et al. 2016), this brain structure has been traditionally ignored in the addiction field. Previous findings from our lab demonstrate the substantial ability of cocaine to induce molecular and structural changes in the cerebellum (Carbo-Gas et al. 2014a, b;Vazquez-Sanroman et al. 2015a, b). Moreover, we have proposed a cerebellum role in cocaineinduced conditioned memory . Our present findings indicate that extended access to cocaine self-administration may dynamically regulate conditions for plasticity in the cerebellum during abstinence. Further research is required to determine whether the degradation of these cerebellar PNNs can prevent relapse after protracted abstinence.