The presence of a social stimulus reduces cocaine seeking in a place preference conditioning paradigm.

BACKGROUND: One challenge in the treatment of substance use disorders is to re-engage the interest toward non-drug-related activities. Among these activities, social interaction has had a prominent role due to its positive influence on treatment outcome. AIMS AND METHODS: Our aim was to study whether the presence of a social stimulus during the cocaine-induced conditioned place preference test was able to reduce the time spent in the drug-paired compartment. For that purpose, mice were trained for four days on a conditioned place preference task with one compartment paired with cocaine and the opposite with saline. On the test day, we introduced an unfamiliar juvenile male mouse into the saline-conditioned compartment (inside a pencil cup) to analyse the animal preference towards the two rewarding stimuli (cocaine vs mouse). Additionally, to discard the possible effect of novelty, as well as the housing condition (social isolation) on social preference, we decided to include a novel object during the test session, as well as perform the same conditioned place preference protocol with a group of animals in social housing conditions. RESULTS: The social stimulus was able to reduce the preference for cocaine and enhance the active interaction with the juvenile mouse (sniffing) compared to the empty pencil cup paired with the drug. The introduction of a novel object during the test session did not reduce the preference for the cocaine-paired compartment, and interestingly, the preference for the social stimulus was independent of the housing condition. c-Fos immunohistochemistry revealed a different pattern of activation based on cocaine-paired conditioning or the presence of social stimulus. CONCLUSIONS: These results suggest that social interaction could constitute a valuable component in the treatment of substance use disorders by reducing the salience of the drug.Plan Propio 2017 – ‘Ayudas para proyectos dirigidos por jóvenes investigadores’, PPIT.UMA.B1.2017/38. Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Pharmacogenetic inhibition of the infralimbic cortex promotes reinstatement of cocaine-context memories in mice.

Funding: PID2020-113806RB-I00, 08-2021-AREA3, B1-2020_06, FPU20/00908, PRE2018-085673, PREDOC_01094, POSTDOC_ 21_00222. II Plan Propio de Investigación, Transferencia y Divulgación Científica de la UMA.Relapse is one of the main problems concerning treatment of cocaine use disorder. It have been suggested that the infralimbic cortex (IL), a division of the medial prefrontal cortex, is involved in extinction and reinstatement of associative memories, including those involving drugs. However, more selective strategies are needed to elucidate the involvement of IL in the long-term maintenance of drug-related maladaptive behaviours. Here, we employed pharmacogenetics to assess the causal role of IL in the reinstatement of a cocaine-induced conditioned place preference (CPP). For this purpose, adult C57BL/6J mice received bilateral intra-IL microinjections of an adeno-associated viral (AAV) vector containing the hM4Di designed receptor (AAV5-CaMKII-hM4Di-mCherry; AAV-hM4Di, n=11) or a control vector (AAV5-CaMKII-mCherry; AAV-control, n=9) prior receiving training in the cocaine-induced CPP model. After habituation, animals received compartment-paired conditioning by increasing doses of cocaine (2-16 mg/kg/day, i.p.) and were tested for cocaine-CPP, after which they were subjected to forced CPP extinction and then re-tested for cocaine-CPP. On day 37 after AAV infusion, mice received Clozapine N-oxide (CNO, 5 mg/kg, i.p.) and 30 min later were tested for cocaine-primed (7.5 mg/kg, i.p.) CPP reinstatement. Ninety minutes after, animals were perfused, and brains dissected. Our results indicated that both groups acquired and subsequently extinguished cocaine-CPP. However, only the AAV-hM4Di group showed a significant preference for the cocaine-paired compartment during the CPP reinstatement test. Immunofluorescence analyses of c-Fos expression in IL revealed a decrease of ~60% in mCherry+/c-Fos+ co-labelling in the AAV-hM4Di group, suggesting reduced IL neural activity during CPP reinstatement. Therefore, our data suggests that the IL plays a causal role in relapse to cocaine-related maladaptive behaviours, highlighting its importance as a potential therapeutic target.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Where to place the rewards? Exploration bias in mice influences performance in the classic hole-board spatial memory test

Author manuscriptThe classic hole-board paradigm (a square arena with 16 holes arranged equidistantly in a 4x4 pattern) assesses both exploration and spatial memory in rodents. For spatial memory training, food rewards are hidden in a fixed set of holes. The animal must not visit (i.e. nose-poke) the holes that are never baited (reference memory; RM) nor re-visit a baited hole within a session (working memory; WM). However, previous exploratory bias may affect performance during reward searching. During habituation sessions with either all holes rewarded or all holes empty, mice intrinsically preferred poking peripheral holes (especially those located in the maze’s corners) over centre holes. During spatial memory training, mice progressively shifted their hole pokes and staying time to the central area that contained hidden rewards, while mice exposed to the empty apparatus still preferred the periphery. A group of pseudotrained mice, for whom rewards were located randomly throughout the maze, also increased their central preference. Furthermore, reward location influenced memory measures. Most repeated pokes (WM-errors) were scored in the locations that were most intrinsically appealing to mice (i.e. the corner and wall baited holes), supporting a strong influence of previous exploratory bias. Regarding RM, finding rewards located in the centre holes, which were initially less preferred, entailed more difficulty and required more trials to learn. This outcome was confirmed by a second experiment that varied the pattern of rewarded holes, as well as the starting positions. Therefore, reward location is a relevant aspect to consider when designing a hole-board memory task.This study was funded by grants from the Spanish Ministry of Economy and Competitiveness (MINECO, Agencia Estatal de Investigación) cofounded by the European Research Development Fund -AEI/FEDER, UE- (PSI2015-73156-JIN to E.C.O.; PSI2017-82604R to L.J.S.) and from University of Malaga (Plan Propio 2017 – ‘Ayudas para proyectos dirigidos por jóvenes investigadores’, PPIT.UMA.B1.2017/38 to P.S.P). Author P.S.P. holds a ‘Juan de la Cierva-formación’ grant from the Spanish Ministry of Economy, Industry and Competitiveness (code: FJCI-2015-23925). Author M.M.P. holds Predoctoral contract from University of Malaga (Plan Propio 2017). Author F.A.G holds Young researchers contract from the University of Malaga co-funded by the Regional Government of Andalusia and the European Social Fund. Author E.C.O holds a ‘Jóvenes Investigadores’ grant (code: PSI2015-73156-JIN) from MINECO-AEI/FEDER, UE

Persistent drug-associated memories coexist with hippocampal-dependent cognitive decline and altered adult hippocampal neurogenesis in mice withdrawn from cocaine.

Aims: Using a new animal model (‘chronic’ cocaine-induced conditioned place preference –CPP- paradigm), this work studied whether the long-term maintenance of cocaine-associated memories was concomitant to cognitive impairment and adult hippocampal neurogenesis (AHN) alterations. Methods: Male c57BL/6J mice were submitted to a CPP task treated either with cocaine (20 mg/kg/day) or saline for 14 days (n=10 per group). Bromodeoxyuridine (BrdU) was administered to label the new hippocampal neurons generated one week after the last cocaine dose. After 28 drug-free days, mice were assessed for the CPP memory and on a battery of emotional and cognitive behavioral tests. After completion of behavior, brains were collected for AHN analysis. Results: In mice treated with cocaine, preference for the cocaine-paired compartment (CPP memory) persisted over time. In addition, the cocaine-withdrawn mice overall displayed normal emotional behavior but they showed hippocampal-dependent cognitive impairment for novelty recognition (object and place) and spatial (reference and working) memory. The number of BrdU+ cells was unaffected, suggesting that cocaine withdrawal did not impair basal AHN. However, the cocaine-withdrawn mice excessively increased the number immature hippocampal neurons (doublecortin+) after behavioral training, in direct correlation with their cognitive performance, probably as a result of effortful learning. Conclusions: The CPP memory induced by cocaine remains unaltered after a prolonged period of abstinence, accompanied by defective acquisition of new learnings. Since the doublecortin+ neurons correlated with better cognitive performance in the cocaine-withdrawn mice, strategies that increase AHN could alleviate neurocognitive deficits induced by cocaine.Plan Propio Universidad de Málaga Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Remote memory of drug experiences coexists with cognitive decline and abnormal adult neurogenesis in an animal model of cocaine-altered cognition

Author manuscriptCocaine addiction is a chronic disorder in which the person loses control over drug use. The past memories of the stimuli associated with the drug are a relevant clinical problem, since they trigger compulsive drug seeking and taking habits. Furthermore, these persistent drug-related memories seemingly coexist with cognitive decline that predicts worse therapeutic output. Here we use a new animal model of cocaine-altered cognition that allowed to observe these events in the same individual and study their relationship. Mice were chronically administered cocaine in a conditioned place preference (CPP) apparatus for 14 days, and control mice received saline. After 28 days of cocaine withdrawal, animals were tested for retrieval of remote drug-associated memory as well as for cognitive performance in a battery of tests, including novel object and place recognition and spatial memory. The cocaine-withdrawn mice showed persistent CPP memory while impaired in the cognitive tasks, displaying deficits in reference memory acquisition and working memory. However, the CPP expression was not associated to the defective cognitive performance, indicating that they were concomitant but independent occurrences. After completion of the experiment, adult hippocampal neurogenesis (AHN) was studied as a relevant neurobiological correlate due to its potential role in both learning and drug addiction. Results suggested a preserved basal AHN in the cocaine-withdrawn mice, but an aberrant learning-induced regulation of these neurons. This paradigm may be useful to investigate maladaptive cognition in drug addiction as well as related therapies.Authors M.C.M-P., S. G-R. and F. A-G. hold predoctoral grants from the Spanish Ministry of Science, Innovation and Universities (FPU17/00276 to M.C.M-P.; FPU18/00941 to S.G-R. and PRE2018-085673 to F.A-G.) and from the University of Málaga (Plan Propio 2017 –‘Contratos predoctorales’ to M.C.M-P.). Author F.A-G held a ‘Garantía Juvenil’ research contract from the University of Málaga co-funded by the Regional Government of Andalusia and the European Social Fund. Author P.S-P. holds a ‘Juan de la Cierva- Formación’ grant (FJCI-2015-23925) from MINECO-AEI/FEDER, UE. Author E.C-O. holds a ‘Jóvenes Investigadores’ grant (code: PSI2015-73156-JIN) from MINECO- AEI/FEDER, UE

The infralimbic cortex and the hippocampal CA1-Subiculum are functionally involved in the extinction of cocaine-context associationes in mice.

Funding: PID2020-113806RB-I00, 08-2021-AREA3, B1-2020_06, PREDOC_01094, PRE2018-085673, FPU20/00908, POSTDOC_ 21_00222. II Plan Propio de Investigación, Transferencia y Divulgación Científica de la Universidad de Málaga.Cocaine abuse is a health and social problem worldwide. Treatment seeking for cocaine use disorder is on the rise, and relapse prevention remains as a primary goal. Interventions based on extinction of cocaine-related associative memories are promising but so far have not been successful. In this sense, further research is needed to elucidate the neurobiological substrates of extinction learning. Here, we aimed to study the neural circuitry involved in extinction of cocaine-context associations in the Conditioned Place Preference (CPP) model. Adult C57BL/6J mice received habituation to the CPP apparatus followed by conditioning with increasing doses of cocaine (2, 4, 8 and 16 mg/kg/day). After testing for CPP acquisition, a group of mice was submitted to four sessions of forced extinction (CPP+EXT, n = 9) while another group was maintained at home-cage (CPP+ACQ, n = 6). Then, both conditions were retested for cocaine-CPP. Ninety minutes later, animals were perfused, and brains collected. Next, we analysed by immunohistochemistry the expression of c-Fos in a variety of addiction-related structures including the medial prefrontal cortex (prelimbic, infralimbic), the striatum (nucleus accumbens, caudate-putamen), the basolateral amygdala and the hippocampus. Our results indicated that both groups acquired cocaine-CPP, but only the CPP+EXT condition ceased to show preference for the cocaine-paired compartment during the CPP retest. Importantly, the CPP+EXT mice showed increased c-Fos expression in the infralimbic cortex (IL), and the hippocampal CA1-subiculum during the CPP retest, with no changes in the other brain areas examined. Multivariate analyses revealed a relationship between IL and CA1-subiculum activity and CPP extinction. This suggest that such structures are functionally involved in retrieval of extinction memory for cocaine-context associations, thus standing out as potential therapeutic targets.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Effects of sequential exposure to physical exercise and cognitive training on hippocampal neurogenesis in mice

AIMS: Physical exercise and cognitive training hippocampal dependent tasks are known to enhance adult hippocampal neurogenesis (AHN). Here we aimed to evaluate the effect of either a moderate-intensity exercise protocol, a working memory task and the combination of both treatments on mice AHN. METHODS: Adult male C57BL6/J mice (N=34) were submitted to a scheduled treadmill exercise protocol for 12 days (EX-groups) or remained at home cage (SED-groups). 24 hours later, animals either were perfused or trained in a spatial learning task in the Water Maze (WM groups) for 8 days while control groups remained at home cage (CAGE groups). Bromodeoxyuridine (BrdU) was injected at the beginning of every experimental procedure to label hippocampal cells that proliferated during the initial exercise sessions. RESULTS: Mice submitted to scheduled exercise showed an increased number of BrdU+ and PCNA+ dentate granule cells (DGCs) in the short but not in the long-term when compared to sedentary groups. Conversely, training in the WM solely reduced the amount of BrdU+ and PCNA+ DGCs compared to CAGE group. However, animals submitted to scheduled exercise and WM training showed increased proliferation/survival of DGCs in the long-term compared to all other groups. CONCLUSIONS: Our data suggests that the combination of moderate-intensity exercise with spatial training has a powerful neurogenic effect in the DG, being a valuable non-pharmacological strategy for the treatment of neurodegenerative diseases associated with impaired AHN. Funding: PSI2017-82604; PRE2018-085673; FPU20/00908; 08-2021-AREA3; B1-2020_06; Posdoc_21_00222; Posdoctoral_a32. I Plan Propio de Investigación, Transferencia y Divulgación Científica de la Universidad de Málaga.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Cognitive impairment and persistent changes in exploration and hyperactivity in mice after withdrawn from chronic cocaine

PSI2017-82604; PRE2018-085673; I Plan Propio de Investigación, Transferencia y Divulgación Científica de la Universidad de MálagaLasting neurobehavioral adaptations such as cognitive decline are induced by chronic cocaine exposure in animal models. However, persistent changes in motor and exploratory responses are rarely reported. In this study, mice were administered a cocaine dose (COC, 20 mg/kg/day) or saline (SAL) repeatedly for 12 consecutive days in their home cage. After 24 days of drug withdrawal, they were submitted to a behavioral test battery to assess motor/exploratory activity and anxiety-like behavior (elevated plus maze and open field tests), behavioral despair (forced swimming test), working and reference memory (spontaneous alternation behavior –SAB- and novel place recognition memory tests). This behavioral assessment was carried out in drug-free conditions and in unfamiliar environments, so no cocaine-associated stimuli were presented. The cocaine-withdrawn mice showed cognitive deficits in spontaneous alternation behavior and place recognition memory. Importantly, they also displayed hyperlocomotion, increased rearing activity and altered exploratory patterns in different tasks. In the forced swimming test, they were more active (struggled/climbed more) when trying to escape from the water albeit showing similar immobility behavior than controls. In conclusion, in addition to cognitive deficits, chronic cocaine may induce lasting changes in psychomotor activation even in unfamiliar environments not associated to the drug. This outcome may be influenced by factors related to exploration, energy or emotionality.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Sequential treadmill exercise and cognitive training synergistically increase adult hippocampal neurogenesis in mice

Combining physical and cognitive training has been suggested to promote further benefits on brain and cognition, which could include synergistic improvement of hippocampal neuroplasticity. In this paper, we investigated whether treadmill exercise followed by a working memory training in the water maze increase adult hippocampal neurogenesis to a greater extent than either treatment alone. Our results revealed that ten days of scheduled running enhance cell proliferation/survival in the short-term as well as performance in the water maze. Moreover, exercised mice that received working memory training displayed more surviving dentate granule cells compared to those untreated or subjected to only one of the treatments. According to these findings, we suggest that combining physical and cognitive stimulation yield synergic effects on adult hippocampal neurogenesis by extending the pool of newly-born cells and subsequently favouring their survival. Future research could take advantage from this non-invasive, multimodal approach to achieve substantial and longer-lasting enhancement in adult hippocampal neurogenesis, which might be relevant for improving cognition in healthy or neurologically impaired conditions.This study received financial support from the Spanish Ministry of Economy and Competitiveness (Agencia Estatal de Investigación, AEI), which was cofounded by the European Regional Development Fund (AEI/FEDER, UE; PSI2017-82604R to L.J.S.); by the Spanish Ministry of Science and Innovation (PID2020-113806RB-I00 to L.J.S); by the Biomedical Research Institute of Málaga (IBIMA; 08-2021-AREA3 to D.L.G.M) and by the University of Málaga (B1-2020_06 to D.L.G-M). Funding for Open Access charge: Universidad de Málaga/CBU

Persistent changes in exploration and hyperactivity coexist with cognitive impairment in mice withdrawn from chronic cocaine

Repeated cocaine exposure induces lasting neurobehavioral adaptations such as cognitive decline in animal models. However, persistent changes in spontaneous –unconditioned- motor and exploratory responses are scarcely reported. In this study, mice were administered with cocaine (20 mg/kg/day) or vehicle for 12 consecutive days. After 24 days of drug abstinence, a behavioral assessment was carried out in drug-free conditions and in unfamiliar environments (i.e. no cocaine-associated cues were presented). The cocaine-withdrawn mice showed cognitive deficits in spontaneous alternation behavior and place recognition memory. Importantly, they also displayed hyperlocomotion, increased rearing activity and altered exploratory patterns in different tasks. In the forced swimming test, they were more active (struggled/climbed more) when trying to escape from the water albeit showing normal immobility behavior. In conclusion, in addition to cognitive deficits, chronic cocaine in rodents may induce long-lasting alterations in exploratory activity and psychomotor activation that are triggered even in absence of drug-related stimuli.This study was funded by grants from the Spanish Ministry of Economy and Competitiveness (MINECO, Agencia Estatal de Investigación –AEI-) cofounded by the European Regional Development Fund-FEDER, UE- (PSI2015–73,156-JIN to E.C–O.; PSI2017–82604R to L.J.S.), RETICS Red de Trastornos Adictivos (ERDF-EU; RD16/0017/0001 to F.R.F.) and University of Málaga (B4: ‘Ayudas para Proyectos Puente’to E.C–O). Funding for open access charge: Universidad de Málaga /CBUA. Authors M.C.M-P., F. A-G. and S. G-R. hold predoctoral grants from the Spanish Ministry of Science, Innovation and Universities (FPU17/00276 to M.C.M-P.; PRE2018–085673 to F.A-G.; and FPU18/00941 to S.G-R.). Author D.L.G.M. holds a postdoctoral grant from University of Málaga (A.3. Plan Propio de Investigación y Transferencia Universidad de Málaga)