PhD Asmae Lguensat

PhD Christian Montanari

Cassandre Vielle

PhD Mohamed Bennis

PhD Saadia Ba-M'hamed

PhD Christelle Baunez

Ph.D René Garcia
email: rene.garcia@univ-amu.fr

Saadia Ba- M'hamed

Repeated cocaine exposure prior to fear conditioning induces persistency of PTSD-like symptoms and enhancement of hippocampal and amygdala cell density in male rats

Keywords: Footshock stress, Rats, Cocaine self-administration, Extinction memory impairment Footshock stress, Rats, Cocaine self-administration, Extinction memory impairment

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Replies to Reviewer # 1

Reviewer # 1 has raised 10 points. Please find below our corresponding replies.

POINT 1. While it is understandable and even desirable to relate the procedures and outcomes in this study to PTSD, these rats have not undergone anything remotely close to trauma. The terms "PTSD" and "trauma" should not appear in the Methods or Results sections at all. Instead, please use a more concrete description of the actual testing conditions. For example, "shock" and "no shock" groups as opposed to "PTSD" and "no PTSD".

REPLY TO POINT 1: As requested by the reviewer, we have replaced 'PTSD' and 'trauma' with either 'fear conditioning' or 'fear related behavior/symptoms'. 'PTSD' and 'no PTSD' groups have been changed to 'shock' and 'no shock' groups, respectively.

POINT 2: The description of results depicted in Figure 8A states that there were no significant effects of "trauma", but the p-value is reported as 0.044 and alpha was set at 0.05. Isn't that significant?

REPLY TO POINT 2: The p-value is indeed significant. We have corrected the text accordingly.

POINT 3: The first sentence on page 19 is confusing. "Furthermore, we showed that a history of cocaine consumption did not induce any sensitization to future cocaine use." Was there an attempt to measure sensitization? If so, what exact comparison is this sentence alluding to? REPLY TO POINT 3: We did not specifically measure sensitization (cocaine-induced behavioral sensitization to the locomotor response). Our intention was to indicate that fear conditioning after cocaine use (20 sessions) did not alter subsequent cocaine use. To avoid confusion, the corresponding sentence has been removed.

Reply to Reviewer # 2

Reviewer # 2 has raised 7 points. Please find below our corresponding replies.

POINT 1: This study only uses male rats. Justification for not using females should be included and this limitation should be acknowledged.

REPLY TO POINT 1: We have indicated in the Material and Methods section that we chose to use only male rats for convenience reasons in our animal facility. We have also acknowledged the fact that despite the hormonal fluctuations associated with the female reproductive cycle, it appears that behavioral data obtained with female rats are not more variable than those of male rats [START_REF] Becker | Female rats are not more variable than male rats: a metaanalysis of neuroscience studies[END_REF].

POINT 2: In the Introduction, an attempt is made to discuss animal models of PTSD, but the authors fail to discuss that fear conditioning is not a model of trauma but rather reflects normal learning to aversive stimuli. The use of fear conditioning in these studies is appropriate but should be put in context and rationales provided to draw similarities to human studies and other animal models of traumatic stress.

REPLY TO POINT 2: The requested changes have been made (see the Introduction section).

POINT 3: The authors fail to report published studies that have investigated the effects of cocaine on stress-related outcomes, including a published study that specifically models cocaine pre-trauma exposure on anxiety-like behaviors following traumatic stress exposure in rats (PMID: 28750215). The authors inaccurately claim that their study is the first to show that cocaine has anxiolytic effects in traumatic stress exposed rats.

REPLY TO POINT 3: Changes has been made as requested by the Reviewer.

POINT 4: In the Discussion, the effects of stress on later cocaine taking are discussed, and the authors conclude that trauma does not change cocaine intake following stress or trauma. A lot of literature that could be referenced is omitted here, and specifically the authors need to consider a few reports that have tested the effects of traumatic-like stress on cocaine taking and related behaviors (PMIDs: 25712697, 26115790, 23664123, 30291225, 29061385, 27181613).

REPLY TO POINT 4: We thank the Reviewer for guiding us to these valuable references.

We have now reviewed them all in the Discussion section of the revised manuscript. POINT 5: The authors claim that hypofunction of the infralimbic cortex facilitates return of PTSD-like symptoms, but only provides 1 reference that this occurs in human or animals.

REPLY TO POINT 5: We have added an example of a human study.

POINT 6: The justification for why hippocampal cell density is increasing is poor and not put in perspective to the many human PTSD-related studies and animal stress-models showing decreases in HC volume and cell density. The published literature needs to be referenced and better integrated into the current findings.

REPLY TO POINT 6:

We have further discussed this point by acknowledging that PTSD is characterized by a reduced hippocampal volume.

POINT 7: The suggestion that cell densities are increased because of inflammatory markers is interesting but overly speculative. In order to justify this claim, the authors should identify the cell type(s) that they are measuring. Identifying neurons form glial will increase the value of the data and provide more evidence that inflammatory response is playing a role in the increased cell density, particularly in the HC.

REPLY TO POINT 7: Unfortunately, we were not able to identify neurons from glial cells. However, we have rephrased our point.

INTRODUCTION

Post-traumatic stress disorder (PTSD) develops after a very stressful, frightening, or distressing event, or a prolonged traumatic experience. Symptoms may improve with treatment, but unfortunately not in everyone [START_REF] Zohar | New insights into secondary prevention in post-traumatic stress disorder[END_REF]. Although it is not clear exactly why some people develop chronic PTSD while others do not, a few possible reasons have been suggested such as co-occurring substance use disorders. Indeed, PTSD is common among people with substance use disorders [START_REF] Mcfarlane | The phenomenology of posttraumatic stress disorders following a natural disaster[END_REF][START_REF] Breslau | Traumatic events and posttraumatic stress disorder in an urban population of young adults[END_REF][START_REF] Saladin | Understanding comorbidity between PTSD and substance use disorders: two preliminary investigations[END_REF]Bremner et al., 1996;[START_REF] Cottler | Gender differences in risk factors for trauma exposure and post-traumatic stress disorder among inner-city drug abusers in and out of treatment[END_REF] those based on footshock stress are particularly popular given their convenience in assessing learning and emotional memory processes following exposure to aversive stimuli [START_REF] Anagnostaras | Computer-assisted behavioral assessment of Pavlovian fear conditioning in mice[END_REF]. Footshock is also a powerful tool to reveal the neurobiological underpinnings of psychiatric disorders in which a strong emotional memory component is present, such as in PTSD [START_REF] Careaga | Understanding posttraumatic stress disorder through fear conditioning, extinction and reconsolidation[END_REF]. However, confusion between paradigms based on footshock-induced long-lasting PTSD-like symptoms and footshock-induced normal conditioned fear without PTSD-like symptoms must be avoided. Indeed, a strong footshock (e.g., a 1.5-mA footshock) has been reported to produce both conditioned fear memory and non-conditioned PTSD-like symptoms (e.g. fear incubation and sensitization, social withdrawal, neophobia and depression-like behavior; Siegmund and Wotjak, 2007). On the other hand, a mild footshock (e.g., a 0.4-mA footshock) is known to induce conditioned fear memory without PTSD-like symptoms [START_REF] Bentefour | Effects of paroxetine on PTSD-like symptoms in mice[END_REF]. Fear conditioning requires gene expression and protein synthesis in both the hippocampus and the basolateral amygdala alongside activation of various metabolic signaling pathways [START_REF] Vianna | Role of the hippocampus and amygdala in the extinction of fear-motivated learning[END_REF]. Interestingly, these requirements are like those for fear extinction [START_REF] Vianna | Role of the hippocampus and amygdala in the extinction of fear-motivated learning[END_REF]. For example, in the hippocampus it has been shown that fear conditioning activates a fear engram, while extinction training inhibits these engram cells and activates a second extinction engram [START_REF] Lacagnina | Distinct hippocampal engrams control extinction and relapse of fear memory[END_REF]. It is therefore possible that a comorbidity with a substance use disorder will potentiate fear engram in the hippocampus and amygdala, which may contribute to resistance to fear extinction. Moreover, we recently found that, in mice, post-footshock exposure to ethanol induces both resistance to avoidance extinction and increases in dendritic density in the prelimbic area of the medial prefrontal cortex (PrL) (Lguensat et al., submitted manuscript). This finding suggests that, in addition to the hippocampus and amygdala, the PrL may also be one of the brain sites displaying changes that correlate with resistance to fear extinction.

Concerning

Using a fear conditioning paradigm in male rats, we here examined the effects of prior cocaine exposure on conditioned fear memory (which may be altered:

MATERIAL AND METHODS

Animals and catheterization surgery

Fifty-three outbred male Lister Hooded rats (Charles River, Saint-Germain-sur-l'Arbresle, France) were used in the present study. Despite the hormonal fluctuations associated with the female reproductive cycle, it appears that behavioral data obtained with female rats are not more variable than those of male rats [START_REF] Becker | Female rats are not more variable than male rats: a metaanalysis of neuroscience studies[END_REF]. However, we chose to use only male rats for convenience reasons in our animal facility. Three rats were eliminated from the study because of their blocked catheters and therefore their measures were not considered. Half of the remaining rats were submitted to self-administration sessions for 20 days while the other half were submitted to self-administration sessions for 52 days (until the end of the experiment).

Animals were maintained in the animal facility, housed in pair, and handled 2 to 3 times per week before surgery. They were submitted to a 12h light/dark cycle (lights on at 7PM) at a constant temperature (22 ± 1•C). The rats had free access to food pellets (Scientific Animal Food & Engineering, Augy, France) and water except during the experiments. All animal care and use conformed to the French regulation (Decree 2013-118) and were approved by the local ethics committee and the Ministère de l'Agriculture (Saisine #3129.01).

Intra-jugular catheter implantation was performed on day 0. Rats were anesthetized using a mixture of ketamine (Imalgene, Merial, Lyon, France) and medetomidine (Domitor, Janssen, Issy-les-Moulineaux, France). Later, the extremity of a catheter, made out of silicone tubing, was inserted in the right jugular vein. The catheter was placed so that it followed a subcutaneous path above the scapula and exited at the level of the back of the rat.

Self-administration of cocaine or NaCl

Apparatus: Self-administration (SA) sessions were conducted in standard SA chambers (operant boxes from Camden Inst and Med Associates, Georgia, VT) placed in sound-attenuating boxes. Each chamber had a central light, two retractable levers (Camden Inst boxes) or two holes (Med Associates boxes) with two cue lights placed above them and a grid floor.

At the beginning of each session, the rats were connected to the chamber tubing, which was protected by a stainless-steel spring and connected to a 10 ml syringe containing either Cocaine or NaCl (0.9%). Each syringe was placed on a pump (Razel Scientific Instruments, St. Albans, VT). Both the chambers and the pumps were run by either a home-built software (Camden Inst boxes) or MedPC IV software (Med Associates boxes).

Procedure: SA sessions began 7 days post-surgery. Twenty-seven animals were assigned to self-administer cocaine, while 23 animals self-administered NaCl. The SA sessions lasted 1 hour per day for 20 or 52 days, according to the group. The aim of continuing cocaine selfadministration for 52 days was to assess any eventual effect of the fear conditioning procedure on the progression of cocaine self-administration.

Before the beginning of the sessions, each rat was taken from its home-cage and had its catheter tested and flushed using an injection of saline solution containing heparin (Heparin Sodium, Sanofi, Paris, France; 5000 U.I/ml) and gentamecin (Pangram, Vibrac, France) to prevent formation of blood clot and infection. The animal was then connected to the chamber tubing, and the 1-hour sessions were launched on the software. When the session started, the central light was turned on. For each rat, an active lever or an active hole was available. When the active lever was pressed or the active nosepoke was performed, the cue light above it lit up for 5 seconds and an intravenous infusion of either cocaine (250µg/90µl) or NaCl (90µl) was delivered (FR1 schedule: every active lever press or active nosepoke resulted in the delivery of an intravenous infusion). A time-out period of 20 seconds following the infusion was imposed, during which a press on the active lever or an active nosepoke was counted as a preservative response and had no consequence. By the end of the session, each rat was disconnected, and the catheter re-tested before returning the animal to its home-cage. The total number of rewards, corresponding to the total number of infusions (either cocaine or NaCl), was considered for the result analysis.

Fear conditioning and assessment of conditioned fear response

Apparatus: Fear conditioning and testing took place in an operant box different from the ones used during the self-administration sessions. The floor of the box was made of a metal grid with bars separated by 1 cm and connected to an electrical shock generator (Med associates, Georgia, VT). A camera was attached to the ceiling of the box to record the behavior of the rat. The box was controlled by a MedPC IV interface that we used to deliver the shocks.

Procedure: Twenty-four hours following the last SA session (day 28), rats were submitted to the first session of footshock exposure. During this session, animals from the shocked groups (NaCl-Shock: n=12 and Coc-Shock: n=14) were placed in the operant box for 180 seconds, where two footshocks (1.5 mA, 2 seconds), were delivered 6 seconds apart. Sixty seconds following the last footshock, rats were removed from the box. Four days later, they were submitted to a second conditioning session. During this session they were put back in the same operant box, and after 120 seconds, received a footshock (2 mA, 10 seconds). The no-shock groups (NaCl-No shock: n=11 and Coc-No shock: n=13) were submitted to the same procedure, but with the shock mechanism inactivated. The situational reminder (SR) sessions for conditioned fear memory testing started the following day. These sessions involved placing the rats in the conditioning box for 30 seconds, on days 34, 40, and 47 (SR1, SR2 and SR3), without any footshock delivery. This procedure, based on short context re-exposures (less than 1 minute/session), has been reported to induce behavioral sensitization (expressed by increased freezing behavior) without causing fear extinction [START_REF] Pynoos | A behavioral animal model of posttraumatic stress disorder featuring repeated exposure to situational reminders[END_REF]Louvart et al, 2006).

Here, conditioned fear memory was assessed by analyzing freezing behavior during each SR session based on the video recordings.

Assessment of the other footshock exposure-related symptoms

The elevated plus-maze test (EPM): This test is designed to assess the anxiety state of rodents.

The apparatus used consisted of four arms of the same size (49.4 cm × 49.4 cm) at 80 cm from the floor and opened on a central square platform (10.5 cm × 10.5 cm). Two open arms (OA) had no walls, while the two other arms were closed (CA) with walls delimiting the corridors.

The maze was illuminated by a 50-watt bulb placed above it.

On day 48, each rat was put in the apparatus for 5 minutes. We initially put the animal 1-Acclimatization phase (duration: 5 min): the rat explored the arena without objects.

2-Exploration of the two identical objects A and B (duration: 5 min):

The rat was placed in the arena with objects A and B and was expected to explore and become familiar with the objects.

3-Exploration of a novel object (duration 5min

): the rat was placed back in the arena with objects A (identical to A used in 2) and C (novel object). The animal was expected to spend more time at exploring the new object. The discrimination ratio was calculated as follows:

𝐷𝑖𝑠𝑐𝑟𝑖𝑚𝑖𝑛𝑎𝑡𝑖𝑜𝑛 𝑟𝑎𝑡𝑖𝑜 = 𝑇𝑖𝑚𝑒 𝑠𝑝𝑒𝑛𝑡 𝑒𝑥𝑝𝑙𝑜𝑟𝑖𝑛𝑔 𝑡ℎ𝑒 𝑛𝑒𝑤 𝑜𝑏𝑗𝑒𝑐𝑡 -T𝑖𝑚𝑒 𝑠𝑝𝑒𝑛𝑡 𝑒𝑥𝑝𝑙𝑜𝑟𝑖𝑛𝑔 𝑡ℎ𝑒 𝑓𝑎𝑚𝑖𝑙𝑎𝑟 𝑜𝑏𝑗𝑒𝑐𝑡 𝑇𝑜𝑡𝑎𝑙 𝑡𝑖𝑚𝑒

Assessment of extinction learning and strength of extinction memory

Extinction learning: For fear extinction training, rats were put back in the conditioning box for a longer duration per day compared to the SR session (10 minutes/day vs. 30 seconds/day). This was done on five consecutive days (from days 51 to 55) without footshock application. Fear behavior (freezing) was assessed during each session. We manually measured freezing time (the time that the animal spends in a fixed position) for at least one second. The cumulative freezing time is expressed in percentage of total time.

Strength of extinction memory:

To test the strength of extinction memory, on day 56 rats were submitted to the same regimen as during the first conditioning session, except that the footshock was applied with a much lower intensity (0.15 mA) which served as footshock priming to facilitate fear return in case of weak extinction memory. The freezing behavior of the rats was evaluated prior to the footshock priming application (day 56) and 24 hours later (day 57).

All behavioral protocol steps are summed up in Figure 1.

Assessment of cortical thickness and cellular density changes

Tissue preparation and Cresyl violet staining: At the end of the protocol, the animals were deeply anaesthetized by an injection of pentobarbital (Dolethal, Vetoquinol, Lure, France, 73 mg/mL, 1.5 mL i.p.). They were then perfused intracardially with PBS solution (pH=7.4) followed by 4% paraforamaldehyde solution in PBS (pH=7.4). The brains were collected and post-fixed overnight in the same paraformaldehyde solution used for the perfusion with stirring at 4°C. These brains were then post-fixed in a 30% sucrose solution overnight. Each brain was then cut in cryostat (Leica Microsystems, Germany) into 50µm sections and mounted on gelatinized slides. These slides were kept in cold until staining.

For Nissl staining, the following baths were used for the slides: a 95° alcohol bath and a water bath before being placed in the Cresyl violet bath until the desired coloration is obtained.

Then the slides were rinsed with water and the dehydration stage was carried out by submerging them into the 70° and then 95° alcohol baths. Finally, the slides were passed through xylene bath and mounted with Eukitt (Sigma-Aldrich).

Image acquisition and quantification: These analyses were carried out on three adjacent brain sections. The regions of interest were determined using the atlas by Paxinos and Waston (1998). The images were taken using a photomicroscope (Olympus CX2) equipped with a camera (Olympus, DP71), connected to a computer with the acquisition software "DP Controller" and analyzed using the ImageJ software (NIH). Analyses were done on 3 adjacent sections/region/rat and by choosing 3 zones of 1 mm2/section. The measurements obtained on the 3 zones were then averaged and the average obtained was considered in statistical analyses.

Statistical analysis

Data was analyzed using GraphPad Prism v8 software. One-way ANOVAs were used to assess the effect of a single parameter, while the effect of independent variables was assessed using two-way ANOVAs (regular and repeated measures) or three-way ANOVAs, followed by Holm-Sidak post-hoc for multiple comparisons. The significance was set at p<0.05.

RESULTS

Self-administration during the first 20 days:

Cocaine vs. NaCl groups: Data showed that rats that self-administered cocaine (Cocaine group) received more rewards (injections) compared to those in the NaCl group (12.04±1.10 vs.

3.70±0.63) (Figure 2). 5B) did not reveal any significant effect of group or block (F (1; 12) =0.020; p=0.887 and F (4; 9) =0.367; p=0.830, respectively). The interaction between these two factors was not significant (F (4; 12) =1.735; p=0.157).

Data from the NaCl sub-groups (NaCl-No shock and NaCl-Shock) were similar regarding the total number of rewards received during the sessions (Figure 6A-B). Two-way repeated measures ANOVA on all sessions (Figure 6A) did not reveal a significant effect of group (F (1; 9) = 0.276, p=0.599), while there was a significant effect of session (F (51; 41) =2.740, p=0.01). The interaction between these factors was not significant (F (51; 41) =0.478, p=0.999).

Two-way ANOVA analysis of data on blocks (Figure 6B) did not reveal a significant effect of group (F (1; 9) =0.358, p=0.552). The effect of block was significant (F (4; 6) =5.545, p=0.001).

The group x block interaction was not significant (F (4; 9) =0.144; p=0.964).

Footshock exposure-related behavioral changes

Conditioned fear response: Before conditioning, all rats exhibited a similar lack of fear response (freezing). However, after the 1.5-mA footshocks, all Shock groups (NaCl-Shock and Coc-Shock) showed freezing behavior, both immediately and during each of the subsequent SR tests, while no freezing was expressed in any of the No-Shock groups (NaCl-No Shock and Coc-No Shock) (Figure 7).

Three-way ANOVA repeated measures (session x shock x self-administration) on these data showed significant effects of session and shock (F (4; 44) =25.68; p<0.001 and F (1; 47) =316.9; p<0.001, respectively). There was no significant effect of self-administration (F (1; 47) =0.389; p=0.533). The analysis of pairwise interactions revealed that the session x shock interaction was significant (F (4; 47) =24.58; p<0.001), while the other interactions were not 2).

Anxiety-like behavior and recognition memory: Data from the EPM test showed that footshock exposure increased the anxiety index in rats that did not self-administer cocaine (the NaCl-Shock group). However, this effect was abolished in rats that self-administered cocaine (Figure 8A). Two-way ANOVA analysis (shock x self-administration) on these data revealed a significant effect of shock (F (1; 47) =4,545; p=0.044) while no effect of self-administration was observed (F (1; 47) =0.171; p=0.196). Moreover, the interaction between the two factors was significant (F (1; 47) =5.376; p=0.024). The Holm-Sidak post-hoc analysis showed that the NaCl-Shock group expressed a significant increase of the anxiety index compared to the NaCl-No shock group (t=2.931, p=0.031). There was no effect of either footshock exposure or cocaine self-administration on locomotor activity (Figure 8B). Two-way ANOVA analysis (shock x self-administration) did not reveal any significant effect of both shock (F (1, 47) =1.452, p=0.234)

and self-administration (F (1, 47) = 0.029, p=0.863). There was no significant interaction between these two factors (F (1; 47) =1.098; p=0.300).

The NOR data showed fear conditioning by itself (NaCl-Shock group) reduced the recognition index. This effect was augmented when cocaine exposure was added (Coc-Shock group) (Figure 8C).

Fear extinction learning and memory

Fear extinction learning: Extinction data showed that the Shock groups expressed a progressive reduction of time spent freezing during extinction training (Figure 9). Three-way ANOVA repeated measures analysis (session x shock x self-administration) indicated a significant effect of session and shock (F (6; 18) = 23.92; p<0.001 and F (1; 24) = 3.476; p=0.003 respectively). There was no significant effect of self-administration (F (1; 24) = 0.396; p=0.529). The analysis of interactions in pairs revealed the significance of the following interactions: session x shock and session x self-administration (F (6; 24) =17.57; p<0.001 and F (6; 24) =3.476; p=0.003), while the shock x self-administration interaction was not significant (F (1; 24) = 0.003; p=0.952). The Extinction memory strength: Data indicate that before footshock priming (with 0.15 mA; D56) all groups displayed low levels of freezing. However, after footshock priming (D57) only the Coc-Shock group showed fear return, indicating a weak extinction memory (Table 3).

Cortical thickness and cellular density assessment

Cortical thickness: The analysis of total cortical thickness in the cingulate cortex (Cg), prelimbic (PrL), and infralimbic (IL) regions of the mPFC did not show any significant difference between the groups (Figure 10A). Three-way ANOVA test (shock x self-administration x mPFC regions) revealed a significant effect of mPFC region (F (2; 21) =118.4; p<0.001), while no significant effect was seen for shock or self-administration (F (3; 20) =1.916; p=0.196 and F (3; 20) =0.147; p=0.171, respectively). No significant interaction between each pair of factors was noted.

The analysis of layer thickness in the Cg did not reveal any difference between the groups for all layers (Figure 10B). Three-way ANOVA test (shock x self-administration x layers) revealed a significant effect of layer (F (2; 21) =213.2; p<0.001), while no significant effect was seen for shock or self-administration (F (3; 20) =0.567; p=0.638 and F (3; 20) =0.903; p=0.345, respectively). No significant interaction between each pair of factors was found.

In the PrL, no significant variation was detected between the layers (Figure 10C).

Three-way ANOVA test (shock x self-administration x layers) demonstrated a significant effect of layer (F (2; 21) =150.7; p<0.001). The other factors (shock and self-administration) were not significant (F (3; 20) =1.436; p=0.242 and F (3; 20) =0.817; p=0.206, respectively). No significant interaction between pairs was seen.

In the IL, as noted for the PrL, no significant variation was found (Figure 10D). Threeway ANOVA test (shock x self-administration x layers) showed a significant effect of layer (F (2; 21) =104.2; p<0.001), while the other factors (shock and self-administration) were not significant (F (3; 20) =1.149; p=0.336 and F (3; 20) =0.355; p=0.556, respectively).

Cell density:

In the medial prefrontal cortex (mPFC): Cell density was evaluated in the three regions of the mPFC (Cg, PrL and IL). Data from the Cg showed that there was no significant difference between the groups (Figure 11A). Two-way ANOVA test (shock x self-administration) performed on these data did not reveal any significant effect of shock or self-administration (F (3; 20) =2.032; p=0.141 and F (3; 20) =1.092; p=0.102, respectively). In the PrL, no significant difference between the groups was recorded (Figure 11B). Two-way ANOVA test did not show any significant effect of shock or self-administration (F (3; 20) =2.178; p=0.122 and F (3; 20) =1.928; p=0.098, respectively). Similarly, in the IL region, no significant difference was observed (Figure 11C). Two-way ANOVA test did not reveal any significant effect of the studied factors (F (3; 20) =0.5945; p=0.625 and F (3; 20) =0.182; p=0.762, respectively).

In the hippocampus: The analysis targeting the hippocampus revealed increases in cell density in CA1, CA2, and CA3 regions for the Coc-PTSD group (Figure 12A).

Two

DISCUSSION

In this study, we first investigated whether a history of cocaine self-administration would interfere with (i) footshock exposure-related behavioral changes (conditioned fear, anxiety-like behavior, and recognition memory impairment), (ii) fear extinction learning and/or memory, and (iii) the progression of cocaine self-administration following footshock exposure. We found that cocaine experience did not alter expression of fear during situational reminders, but it reduced anxiety-like behavior and potentiated cognitive decline. Behavioral data also show that cocaine facilitated the fear priming effect, suggesting difficulty to recover from a core PTSDlike symptom. Moreover, cocaine intake remained unchanged after footshock exposure.

Examination of cortical thickness and cellular density in structures of interest (i.e., the hippocampus, amygdala and mPFC) revealed that pre-fear conditioning cocaine intake provoked an elevated cellular density in the hippocampal CA1, CA2 and CA3 regions and the BLA.

Behavioral data

Cocaine self-administration: Several hypotheses have been developed to explain the interactions between substance use disorders and PTSD. The most investigated is the selfmedication hypothesis, according to which individuals with PTSD use psychoactive substances in an attempt to cope with their PTSD symptoms [START_REF] Khantzian | The self medication hypothesis of addictive disorders: Focus on heroin and cocaine dependence[END_REF][START_REF] Brown | Substance abuse and post-traumatic stress disorder comorbidity[END_REF].

Another interesting but less studied explanation for these behaviors is the high risk/susceptibility hypothesis, according to which, a prior history of substance use increases the risk of exposure to dangerous and threatening situations, and therefore increasing their risk of developing PTSD (Cordovilla-Guardia et al., 2014). Additionally, drug users become more susceptible to PTSD following trauma exposure [START_REF] Chilcoat | Investigations of Causal pathways between PTSD and Drug Use Disorders[END_REF]. Based on the latter hypothesis, the present rodent study tested the effect of an extended (20 session), but moderate (1h/session), intake of cocaine on footshock exposure-associated behavioral changes and fear extinction learning and memory.

On a separate group of animals, we were interested in assessing the evolution of cocaine self-administration in rats after footshock exposure. Our data did not reveal any effect of footshock stress on cocaine consumption. Our findings agree with multiple reports that show short access to cocaine, self-administered through a FR1 schedule, remains unchanged after [START_REF] Kubiak | The effects of PTSD on treatment adherence, drug relapse, and criminal recidivism in a sample of incarcerated men and women[END_REF]. Taken together, one can conclude that exposure to stress is less likely to induce any increase in cocaine consumption.

Traumatic fear during situational reminders. Reexperiencing trauma is a hallmark symptom of PTSD [START_REF] Ehlers | A cognitive model of posttraumatic stress disorder[END_REF]. It can occur intrusively (such as post-traumatic flashbacks)

or during exposure to conditioned stimuli (such as places and people). In rodent experiments, the model used for reexperiencing corresponds to brief re-exposure to the trauma context without further footshock application [START_REF] Pynoos | A behavioral animal model of posttraumatic stress disorder featuring repeated exposure to situational reminders[END_REF][START_REF] Louvart | Long-term behavioural alterations in female rats after a single intense footshock followed by situational reminders[END_REF]. This procedure is also known to maintain, rather than decrease, the expression of fear over time [START_REF] Korem | Cannabinoids prevent the effects of a footshock followed by situational reminders on emotional processing[END_REF]. Here, we confirmed the maintenance of traumatic fear over sessions and showed that this fear was not altered by the experience of cocaine. According to Bauer (2014), in order to detect the effect of an addictive substance on fear learning or memory, it must be administered immediately before or after training, respectively. In our case, the last cocaine self-administration session took place hours before the first footshock exposure session, explaining why no effect of cocaine was observed on traumatic fear. [START_REF] Zheng | Prefrontal high-frequency stimulation prevents sub-conditioning procedure-provoked, but not acute stress-provoked, reemergence of extinguished fear[END_REF][START_REF] Bentefour | Paroxetine treatment, following behavioral suppression of PTSD-like symptoms in mice, prevents relapse by activating the infralimbic cortex[END_REF]. Interestingly, this procedure can also provoke the relapse of PTSD-like symptoms in mice [START_REF] Bentefour | Paroxetine treatment, following behavioral suppression of PTSD-like symptoms in mice, prevents relapse by activating the infralimbic cortex[END_REF][START_REF] Bentefour | High-frequency stimulation of the infralimbic cortex, following behavioral suppression of PTSD-like symptoms, prevents symptom relapse in mice[END_REF]. Here, we noticed that extinction of traumatic fear occurred in rats exposed to cocaine. However, footshock priming led to a return of traumatic fear in these rats. We have recently reported a similar finding with the use of alcohol In summary, the repeated use of cocaine before footshock stress induction is implicated in amplification of stress-associated impairment of recognition memory and weakening of extinction memory. Our data also show that the repeated use of cocaine before footshock stress induction increased cell density in the hippocampus and amygdala. According to our interpretation, this reflects increases in neuronal and/or glial cells, which may contribute to the observed reactivation of conditioned fear memory and, by extrapolation, the impaired recovery from PTSD. Title: Repeated cocaine exposure prior to fear conditioning induces persistency of PTSD-like symptoms and enhancement of hippocampal and amygdala cell density in male rats.

(
Dear Doctor Shapiro, My colleagues and I would like to thank the Reviewers for their useful comments, which we have fully considered (all changes are in red in the revised manuscript).

We would like to thank you for giving us this chance to improve our manuscript.

We hope that the manuscript is now suitable for publication in Brain Structure and Function.

We look forward to your decision.

Sincerely,

Prof. Rene Garcia

Cover letter

.

  Approximately half of individuals seeking treatment for substance use disorders meet current criteria for PTSD (Berenz and Coffey, 2012). For example, up to 43% of cocaine-dependent individuals meet criteria for lifetime PTSD (Back et al., 2000). In most cases, one disorder worsens the other, increasing the possibility of relapse and decreasing the chances of recovery (Berenz and Coffey, 2012; McGovern et al., 2015). For instance, cocaine addicts with comorbid PTSD are more resistant to treatment than those without a dual diagnosis (Coffey et al., 2002). An explanation for PTSD treatment failure in cocaine users could be drug-induced stronger fear conditioning and impaired extinction learning (Kaag et al., 2016). Animal models based on cocaine self-administration and fear conditioning/extinction may be useful for improving our understanding of (i) how PTSD and substance use disorders interact and (ii) which biological disruptions contribute to resistance to treatment. Most studies carried out so far have investigated how PTSD could affect vulnerability to addiction (María-Ríos and Morrow, 2020), while studies investigating the effects of drug use on PTSD-like phenotype mostly included drug injections (Gordon and Rosen 1999; Blanchard and Blanchard 1999; Burke et al. 2006; Wood et al. 2007). However, none of these studies investigated how regular use of cocaine (in the form of cocaine self-administration) could affect vulnerability to PTSD-like symptoms. In addition, while many animal models of PTSD not based on footshock stress are commonly used [including predator stress (Adamec et al., 2006), single prolonged stress (Librezon et al., 1997), underwater trauma (Richter-Levin, 1998)],

  fear conditioning, it is known that in PTSD the fear responses triggered by the initial trauma are repetitively reexperienced, often through flashbacks and recurring nightmares (Goswami et al., 2013). Regarding extinction, PTSD patients have been reported to be deficient at learning that stimuli previously associated with adverse outcomes no longer presents a threat (Milad et al., 2008; Jovanovic et al., 2012). These findings suggest that deficits in fear extinction underlie resistance to PTSD recovery (Wessa & Flor, 2007; Fenster et al., 2018). Based on this idea, we have recently found that ethanol exposure does not interfere with avoidance, which is a core symptom of PTSD (Sheynin et al., 2017), but impairs avoidance extinction (Lguensat et al., submitted manuscript). More importantly, we have previously demonstrated with the same mouse model (Bentefour et al, 2015) that avoidance extinction training suppresses not only avoidance behavior but all other PTSD-like symptoms (Bentefour et al, 2016). In other words, resistance to extinction indicates impairment of recovery from PTSD-like symptoms.

  Wood et al., 2007), postfootshock anxiety-like behavior (which may not be altered: Lisieski and Perrine, 2017), postfootshock recognition memory (which may be altered: Mottarlini et al., 2020) and fear extinction (which may also be altered: Burke et al., 2006). To identify associated brain changes, we examined cortical thickness in the medial prefrontal cortex and cellular density changes in the hippocampal CA1, CA2, and CA3 regions; basolateral amygdala; and medial prefrontal cortex.

  at the junction of the four arms of the maze, facing an open arm. The animal was free to explore the maze. The animal's behavior was recorded to determine the number of arm entries and time spent in each open arm. The anxiety index was calculated as follows (Rao and Sadananda, Locomotor activity measures were obtained using ToxTrac software (Rodriguez et al., 2017, 2018). The Novel Object Recognition test (NOR): The NOR test was done in a circular arena (diameter: 65cm) and performed over 2 days. During the first day (day 49), the rats freely explored the arena for 15 minutes. The next day (day 50), they underwent the NOR procedure, which consisted of placing each animal in the arena with two objects A and B. The procedure consisted of 3 phases separated by 15 minutes each (Eagle et al., 2013):

  significant (session x self-administration: F (4; 47) =0.467; p=0.474 and shock x selfadministration: F (1; 47) =0.085; p=0.420). The Holm-Sidak post-hoc test revealed that the NaCl-Shock and Coc-Shock groups showed a significantly longer freezing time compared to the NaCl-No shock group and to the Coc-No shock group (Table

Holm-

  Sidak post-hoc test showed that between Ext 1 (D51) and Ext 4 (D54), freezing time expressed by the NaCl-Shock and Coc-Shock groups significantly exceeded fear response expressed by the NaCl-No shock and Coc-No shock groups. During Ext 5 (D55), only rats in the Coc-Shock group continued to show a significant difference compared to the previous groups and to the NaCl-Shock group.

  predator-scent stress[START_REF] Hadad | Conditioned stress prevents cue-primed cocaine reinstatement only in stress-responsive rats[END_REF] or single prolonged stress (SPS)[START_REF] Eagle | Single prolonged stress effects on sensitization to cocaine and cocaine self-administration in rats[END_REF][START_REF] Enman | Anhedonia, Reduced Cocaine Reward, and Dopamine Dysfunction in a Rat Model of Posttraumatic Stress Disorder[END_REF]. In contrast, modified SPS reduced cocaine self-administration only during initial acquisition[START_REF] Hofford | Modified single prolonged stress reduces cocaine self-administration during acquisition regardless of rearing environment[END_REF], suggesting that the type of stress may play a role in determining how drug self-administration evolves. In addition to the type of stress, interindividual differences in cocaine self-administration might be detected within stressexposed animals. In this regard, Schwendt and colleagues (2018) reported that stress-resilient animals self-administered less cocaine compared to stress-susceptible and control animals. This suggests that there might be interindividual differences in cocaine self-administration within shocked animals of our study, and that we need a larger sample size, which would allow us to distinguish between stress-susceptible and stress-resilient animals. Beyond regular selfadministration procedures, footshock stress seems to have no effect on drug-seeking behavior, although it promotes relapse[START_REF] Pizzimenti | Persistent effects of acute stress on fear and drug-seeking in a novel model of the comorbidity between post-traumatic stress disorder and addiction[END_REF]. Indeed, many studies in rodents have highlighted the phenomenon of relapse to substances under the effect of stress[START_REF] Wang | Stress or drug priming induces reinstatement of extinguished conditioned place preference[END_REF][START_REF] Shaham | The reinstatement model of drug relapse: history, methodology and major findings[END_REF]. However, the effect of the latter on substance use and substanceseeking behavior is less clear[START_REF] Liu | Additive effect of stress and drug cues on reinstatement of ethanol seeking: Exacerbation by history of dependence and role of concurrent activation of corticotropin-releasing factor and opioid mechanisms[END_REF][START_REF] Mcfarland | Limbic and Motor Circuitry Underlying Footshock-Induced Reinstatement of Cocaine-Seeking Behavior[END_REF]. In humans, it has been shown individuals suffering from comorbid PTSD and substance use disorder do not differ in terms of consumption from individuals suffering from substance abuse disorder alone[START_REF] Brown | Substance use disorder and posttraumatic stress disorder comorbidity: Addiction and psychiatric treatment rates[END_REF][START_REF] Eggleston | Suicidality, aggression, and other treatment considerations among pregnant, substance-dependent women with posttraumatic stress disorder[END_REF], but they are more likely to relapse after long periods of abstinence (

  Anxiety-like behavior. High anxiety levels are also a hallmark of PTSD[START_REF] Pitman | Biological studies of post-traumatic stress disorder[END_REF].As in humans, studies with rodent models have revealed exaggerated levels of anxiety among other PTSD-like symptoms[START_REF] Blanchard | Fear conditioning in posttraumatic stress disorder: evidence for delayed extinction of autonomic, experiential, and behavioural responses[END_REF][START_REF] Pynoos | A behavioral animal model of posttraumatic stress disorder featuring repeated exposure to situational reminders[END_REF][START_REF] Bentefour | Effects of paroxetine on PTSD-like symptoms in mice[END_REF][START_REF] Lguensat | Susceptibility and Resilience to PTSD-Like Symptoms in Mice Are Associated with Opposite Dendritic Changes in the Prelimbic and Infralimbic Cortices Following Trauma[END_REF]. In the current study, we observed this effect and found that it was abolished in rats with a history of cocaine self-administration. Previous rodent studies, which were not based on stress, have provided contradictory results, with some showing an increase (Basso et al., 1999; Perrine et al., 2008; Aujla et al., 2008) or a decrease (Erhardt et al., 2006; Mueller et al., 2008; Lisieski et al., 2019) in anxiety levels, and others indicating any of these effects (Sarnyai et al., 1995 ; Mantsch et al., 2008). In a study modeling cocaine pretrauma exposure, Lisieski and Perrine, (2017) reported that the drug decreased anxiety while increasing the overall locomotor activity and exploration of the animal. In contrast, our data show that the observed effect of cocaine on stress-induced anxiety is related to a decreased anxiety index, without affecting activity, indicating a specific attenuation of anxiety. Cognitive impairment. PTSD is associated with deficits in various cognitive domains including attention, processing speed, memory, and executive functioning (Morgan et al., 2006; Poljac et al., 2011; Stricker et al., 2017). Similarly, impairment of cognitive abilities are observed in cocaine users (Kumar et al., 2019). Regarding rodent models trauma is known to induce cognitive function impairment (for example: recognition memory impairment: Eagle et al., 2013; Perez-Urrutia et al., 2017; alterations in cognitive flexibility: George et al., 2015). Furthermore, cocaine intake alone has been reported to alter cognitive functions (Briand et al., 2009; Gobin et al., 2019; Preston et al., 2019). This effect is long-lasting. For instance, Briandet al., (2009) showed that extended access to cocaine produced deficits in recognition memory that persisted for at least 2 weeks after the cessation of drug use. In the present study, we found effects of trauma alone and cocaine use alone on recognition memory. The cognitive impairment was amplified in footshock exposed rats with a history of cocaine, suggesting that the presence of both experiences (cocaine use and stress) leads to a more potentiated cognitive decline. It has been shown that the hippocampus is one of the key structures involved in object recognition processes[START_REF] Reger | Ontogeny of Rat Recognition Memory measured by the novel object recognition task[END_REF]. This led us to speculate that, in our study, cocaine and footshock stress probably induced a cumulative dysfunction in the hippocampus. Suppression of conditioned fear. One of the core problems in PTSD is the reduced ability to suppress fear under safe conditions (Davidson et al., 2004; Jovanovic et al., 2011). One way to test this deficiency is to conduct fear extinction experiments with PTSD patients. Such studies have shown either impaired extinction learning (Blechert et al., 2007) or normal extinction learning, but impaired extinction memory (Milad et al., 2008). Different results may depend upon study conditions. In rodent studies, behavioral blockade of extinction memory recall is commonly obtained using the footshock priming procedure (Deschaux et al., 2011;

Figure 1 .

 1 Figure 1. The behavioral experiments timeline.

Figure 2 .

 2 Figure 2. The mean number of cocaine and/or NaCl rewards (injections) per session and per

Figure 3 .

 3 Figure 3. The mean number of rewards (cocaine injections) per session and per blocks of

Figure 4 .

 4 Figure 4. The mean number of rewards (NaCl injections) per session and per block of sessions.

Figure 5 .

 5 Figure 5. The mean number of rewards (cocaine injections) per session and per block of

Figure 6 .Figure 11 .

 611 Figure 6. The mean number of rewards (NaCl injections) per session and per block of sessions.

Figure 12 .Figure 13 .

 1213 Figure 12. Cell density measures in different regions of the hippocampus. A-Representative

Figure 1 FigureFigure 5 6

 15 Figure 1

  Figure 6:

  On one hand, the Holm-Sidak post-hoc test revealed, on the one hand, that the NaCl-Shock group had a reduced recognition index compared to the NaCl-No shock group (t=3.199, p=0.014). On the other hand, the Coc-Shock group expressed a

	The two-factor ANOVA test (shock x self-administration) indicated
	significant effects of shock and self-administration (F (1; 47) =32.14; p<0.001 and F (1; 47) =6.571;
	p=0.013, respectively). However, the interaction between these two factors was not significant
	(F (1; 47) =0.934; p=0.338).

significantly reduced recognition index compared to the NaCl-No shock and Coc-No shock groups (t=5.803, p<0.001 and t=4.893, p<0.001).

(Lguensat et al., submitted).

	In humans, PTSD is associated with prefrontal
	cortical dysfunction (Zubieta et al., 1999; Bremner et al., 2000) and antidepressant-induced
	activation of the prefrontal cortex prevents PTSD relapse (

Davidson et al., 2001; Martenyi et al., 2002; Martenyi and Soldarenkova 2006; Yuan et al., 2018).

  Similarly, hypofunction of the infralimbic area of the mPFC facilitates the return of PTSD-like symptoms in animals

(Bentefour et al., 2016, 2018).

  This leads us to suggest, based on our findings, that cocaine and footshock stress might induce dysfunction in this prefrontal area, reducing the ability of inputs from the prefrontal cortex to inhibit amygdala activity, knowing that increased activation of the amygdala characterizes PTSD state[START_REF] Nutt | Structural and functional brain changes in posttraumatic stress disorder[END_REF]).

	Brain data
	Cortical measures: thickness and cellular density. In humans, a prolonged history of cocaine
	use has been reported to be associated with reduced gray matter volume in different cortical
	regions, including the prefrontal cortex (

Sim et al., 2007; Makris et al., 2008; Crunelle et al.,

	2014). A similar result has also been reported in rats (Cannella et al., 2018). Here, we did not
	observe any significant change in cortical thickness or cell density in the mPFC of rats exposed
	to cocaine alone or to cocaine and trauma. In the study of Cannella and colleagues (2018), rats
	were separated into two groups showing either addict-like or non-addict-like behavior based on
	their inability to refrain from drug-seeking and taking, high motivation for the drug, and
	maintenance of drug use despite negative consequences. Cortical changes were found only in
	rats displaying addict-life behavior (Cannella et al., 2018). Although in human studies
	structural changes have been reported in non-dependent cocaine users (

Mackey et al., 2014), it

  is more likely that the lack of cocaine effect in the present study resulted from our protocol of cocaine intake, which did not model cocaine addiction. Consistent with a previous study in rats[START_REF] Gao | Glutamate and GABA imbalance promotes neuronal apoptosis in hippocampus after stress[END_REF], we did not observe any effect of footshock exposure on thickness or cell density. However, we cannot conclude that our experiment protocol, which failed to induce alteration in the mPFC cytoarchitecture, also failed to induce alteration in the mPFC functioning. This is, in part, corroborated by our behavioral data revealing that a history of cocaine use can facilitate the return of extinguished fear, which has been observed in case of hypofunction of the mPFC (e.

	Contradictory data have been obtained regarding cortical changes in PTSD, with some
	studies indicating decreased thickness (e.g Kühn & Gallinat, 2013; O'Doherty et al., 2015)
	and activity (Hughes and Shin, 2011), while others showing no changes (Landré et al., 2010;
	Rinne-Albers et al., 2020) or an increase in microglial cell number and density (Smith et al.,
	2019).

g. in humans: Milad et al., 2005; e.g. in mice: Bentefour et al., 2016, 2018).

	Cellular density in the hippocampus and amygdala. Studies of patients with PTSD have shown
	structural abnormalities in limbic areas, including two key regions: the hippocampus and the
	amygdala (

Shin et al., 2006). Reduced hippocampal volume is frequently observed in PTSD (Bremner et al., 1995; Gurvits et al., 1996; Karl et al., 2006).

  When assessing the effect of stress on hippocampal integrity, the volume reduction could be explained by the loss of dendrites and their synapses (

Kassem et al., 2013; O'Doherty et al., 2015).

	However, some
	reports point out the possibility that smaller hippocampal volume is a vulnerability factor rather
	than a result of exposure to trauma (Gilberston et al., 2002; Rubin et al., 2016). On a cellular
	level, it has been reported that hippocampal pyramidal neurons are vulnerable to damage caused
	by stress through the action of corticosterone in rodents (e.

g McEwen et al., 1986; Stein- Behrens et al., 1994) and

  cortisol in primates including humans (e.g Uno

et al., 1989; Wingenfeld and Wolf, 2014).

  The damage is mainly represented as dendritic spine loss

(Radley et al., 2006; Clarke et al., 2018; Smith et al., 2019) and

  /or microglia activation and cell changes (

Milior et al., 2016; Smith et al., 2019; Li et al., 2021).

  The latter could eventually have repercussion on cell density leading to its increase.

	Concerning the amygdala volume, the findings are more controversial, with some
	studies showing no significant changes (e.g. Woon and Hedges, 2009), whereas others indicate
	significantly smaller left amygdala volumes in with PTSD patients as compared to both healthy
	and trauma-exposed controls (e.g. Karl et al., 2006). At a cellular level, rodent models of PTSD
	indicate that stress affects the cytoarchitecture of the amygdala either greatly (e.g.

Cohen et al., 2014

  It is therefore possible that cocaine self-administration

	experience facilitated reactivation of fear memories by increasing fear engram activity in the
	hippocampus and amygdala. Second, it is well known that the neuroimmune response to various
	threats (including stress and drug use) operates through neuroinflammation and manifests as
	proliferation as well as morphological and functional changes of microglia and astrocytes

) or poorly (e.g.

[START_REF] Smith | Microglial cell hyper-ramification and neuronal dendritic spine loss in the hippocampus and medial prefrontal cortex in a mouse model of PTSD[END_REF]

. In the present rodent study, we found that footshock exposure induced an increase in cell density in the amygdala (in the BLA), but not in the hippocampus. However, in stressed rats previously exposed to cocaine self-administration, there was an increase in cell density in the hippocampus CA1, CA2, and CA3 regions and amplification of the BLA change. Changes in these structures could have resulted from two major causes. First, we observed that extinction memory was weak in rats with a history of cocaine intake. It has been demonstrated that activity of hippocampal and BLA ensembles regulates the expression of extinction memory, with high activity of fear engrams facilitating the return of the extinguished fear

[START_REF] Lacagnina | Distinct hippocampal engrams control extinction and relapse of fear memory[END_REF]

. Activation of the BLA has also been demonstrated to be essential for both persistence of newly acquired and reactivated fear memories

[START_REF] Nakayama | Late Arc/Arg3.1 expression in the basolateral amygdala is essential for persistence of newly-acquired and reactivated contextual fear memories[END_REF]

.

[START_REF] Ransohoff | Innate immunity in the central nervous system Find the latest version : Review series Innate immunity in the central nervous system[END_REF]

. PTSD is known to be associated with neuroinflammation through the expression of proinflammatory cytokines (

Hammad et al., 2012; Lindqvist et al., 2014; Dennis et al., 2016; Wang et al., 2019).

  Rodent models of PTSD have also provided similar observations (

Aga-Mizrachi et al., 2014; Wohleb et al., 2014).

  Furthermore, PTSD-associated changes in proinflammatory cytokines were observed in the hippocampus (

Peng et al., 2013; Lee et al., 2016) and amygdala (Wilson et al., 2013).

  Furthermore, microglial cell changes and hyper-ramification have been recently reported in the hippocampus (

Smith et al., 2019; Li et al., 2021).

  Therefore, we could conclude a possible link between stress-induced microglial changes in these regions and an eventual elevated cell density. However, elevated cell density in the hippocampus was not found because of footshock exposure itself, but because of cocaine self-administration experience. In this context, it is interesting to note that the repeated use of cocaine promotes inflammation and activation of microglia in both humans (

Zaparte et al., 2019) and rodents (López-Pedrajas et al., 2015; Cotto et al., 2018; Burkovetskaya et al., 2020

). Moreover, since drug use induces an excessive release of dopamine and glutamate, these neurotransmitters binding to receptors expressed on microglia and astrocytes, could amplify inflammatory signaling via the additional release of cytokines. As a result, there is greater activation of the microglia (Kohno et al., 2019) and potentially, more cell density, as seen here.

Table 1 :

 1 statistical values of the comparison between Cocaine and NaCl groups

		Comparison : Cocaine vs. NaCl
		NaCl	Cocaine
	Session			Post-hoc (NaCl vs. Cocaine)
		(mean ±SEM)	(mean ±SEM)
				t	p
	1	3.13±0.39	11.25± 0.92	8.113	***
	2	4.26±0.61	10.77± 0.92	8.457	***
	3	3.51±0.48	12.14 ±1.08	7.454	***
	4	3.34±0.47	12.88±1.11	8.172	***
	5	3.78±0.23	12.22±1.01	9.172	***
	6	3.13±0.41	12.22±1.03	8.822	***
	7	4.21±0.82	12±0.89	7.754	***
	8	3.17±0.54	12.51±1.02	6.791	***
	9	4.86±1.32	12.37±1.11	6.201	**
	10	4.78±0.85	12.37±0.92	8.249	***
	11	3.65±0.61	11.18±0.82	7.257	***
	12	3.56±0.55	11.14±1.00	8.254	***
	13	3.26±0.50	13.66±1.26	5.217	***
	14	3.21±0.50	12.22±0.99	6.575	***
	15	3.52±0.55	12.33±1.13	7.531	***
	16	3.60±0.68	12.37±0.96	8.567	***
	17	3.82±0.69	11.81±0.99	7.263	***
	18	2.95±0.51	11.96±1.02	7.600	***
	19	4.13±0.67	11.74±1.00	6.319	***
	20	3.95±0.65	11.77±0.93	6.172	***

Table

Table 2 :

 2 Statistical data of the different comparisons made between groups during different situational reminder (SR) sessions.

				Post-hoc	Post-hoc	Post-hoc	Post-hoc
				(NaCl-		(NaCl-		(Coc-	(Coc-
		NaCl-Shock	Coc-Shock	Shock vs.	Shock vs.	Shock vs.	Shock vs.
	Session
		(mean±SEM)	(mean±SEM)	NaCl-No	Coc-No	NaCl-No	Coc-No
				shock)		shock)		shock)	shock)
				t	p	t	p	t	p	t	p
	Before	0.278±0.278	0.331±0.184	0.33 ns	0.26 ns	0.08 ns	0.15 ns
	footshocks (D28)
	After footshocks	25.980±4.300	16.533±3.411	5.71 *** 6.11 ### 3.51 **	3.83 ###
	(D28)
	SR1 (D34)	28.817±5.212	29.538±5.753	6.65 *** 7.03 ### 6.95 *** 7.36 ###
	SR2 (D40)	33.992±3.306	37.787±4.862	8.12 *** 8.25 ### 9.19 *** 9.38 ###
	SR3 (D47)	35.612±4.493	31.305±2.755	8.21 *** 8.10 ### 7.31 *** 7.17 ###

Table 3 :

 3 Different comparisons made during extinction and relapse sessions.

				Post-hoc	Post-hoc	Post-hoc	Post-hoc
				(NaCl-		(NaCl-		(Coc-	(Coc-Shock
		NaCl-Shock	Coc-Shock	Shock	vs.	Shock vs.	Shock vs.	vs.	Coc-No
	Session
		(mean±SEM)	(mean±SEM)	NaCl-No	Coc-No	NaCl-No	Shock)
				shock)		Shock)		Shock)
				t	p	t	p	t	p	t	p
	Ext1 (D51)	46.43±4.05	37.32±5.00	10.19 ***	8.33 ### 8.18 ***	10.14 ###
	Ext2 (D52)	42.25±5.21	28.93±4.80	9.27 ***	7.27 ### 7.01 ***	9.83 ###
	Ext3 (D53)	26.56±3.07	25.37±5.58	9.82 ***	8.03 ### 6.82 ***	7.92 ###
	Ext4 (D54)	21.18±1.96	17.28±3.88	1.82 ns	5.92 ##	5.02 *	6.19 #
	Ext5 (D55)	5.62±1.29	16.39±4.78	1.38 ns	2.01 ns	4.91 *	5.29 #
	Before FS	6.62±3.69	10.85±1.97	1.02 ns	0.98 ns	1.81 ns	0.87 ns
	priming (D56)
	After FS priming	0.00±0.00	17.91±2.855	2.11 ns	1.23 ns	0.23 **	0.79 ##
	(D57)

Funding information

This work was supported by a Marie Curie International Research Staff Exchange Scheme Fellowship within the 7th European Community Framework Programme (PIRSES-GA-2012-318997) and the foreign doctoral student support grant of the City of Marseille, France.

Compliance with ethical standards

All experimental procedures were conducted in agreement with the French regulation (Decree 2013-118) and were approved by local ethic committee and the Ministère de l'Agriculture (Saisine #3129.01).

Conflict of interest

The authors declare that they have no conflict of interest.

(session 1-20) and after PTSD induction (session 21-52). B-Block analysis of the number of rewards during different blocks of the 52 sessions. Bars represent mean ± SEM.