参见作者原研究论文

本实验方案简略版
Feb 2020

本文章节


 

An Operant Conditioning Model Combined with a Chemogenetic Approach to Study the Neurobiology of Food Addiction in Mice
操作式条件反射模型结合化学遗传学方法研究小鼠食物成瘾的神经生物学机制   

引用 收藏 提问与回复 分享您的反馈 Cited by

Abstract

The study of food addiction comprises 3 hallmarks that include the persistence to response without an outcome, the strong motivation for palatable food, and the loss of inhibitory control over food intake that leads to compulsive behavior in addicted individuals. The complex multifactorial nature of this disorder and the unknown neurobiological mechanistic correlation explains the lack of effective treatments. Our operant conditioning model allows deciphering why some individuals are vulnerable and develop food addiction while others are resilient and do not. It is a translational approach since it is based on the Diagnostic and Statistical Manual of Mental Disorders 5th edition (DSM-5) and the Yale Food Addiction Scale (YFAS 2.0). This model allows to evaluate the addiction criteria in 2 time-points at an early and a late period by grouping them into 1) persistence to response during a period of non-availability of food, 2) motivation for food with a progressive ratio, and 3) compulsivity when the reward is associated with a punishment such as an electric foot-shock. The advantage of this model is that it allows us to measure 4 phenotypic traits suggested as predisposing factors related to vulnerability to addiction. Also, it is possible to evaluate the long food addiction mouse model with mice genetically modified. Importantly, the novelty of this protocol is the adaptation of this food addiction model to a short protocol to evaluate genetic manipulations targeting specific brain circuitries by using a chemogenetic approach that could promote the rapid development of this addictive behavior. These adaptations lead to a short food addiction mouse protocol, in which mice follow the same behavioral procedure of the early period in the long food addiction protocol with some variations due to the surgical viral vector injection. To our knowledge, there is no paradigm in mice allowing us to study the combination of such a robust behavioral approach that allows uncovering the neurobiology of food addiction at the brain circuit level. We can study using this protocol if modifying the excitability of a specific brain network confers resilience or vulnerability to developing food addiction. Understanding these neurobiological mechanisms is expected to help to find novel and efficient interventions to battle food addiction.

Keywords: Food addiction (食物成瘾), Vulnerability (弱点), Resilience (恢复力), Operant conditioning (操作式条件反射), Viral vector approach (病毒载体法), Chocolate-flavored pellets (巧克力风味球), Compulsivity (强迫性), Impulsivity (冲动型)

Background

In the last years, food addiction has gained attention due to the increasing prevalence worldwide (19.9 %) and currently represents a high cost to the individual and the society without any effective treatment available (Pursey et al., 2014). The current diagnosis is performed by a recently validated tool, the Yale Food Addiction Scale 2.0 (YFAS 2.0). This instrument is based on the criteria applied in the 5th edition of the Statistical Manual of Mental Disorders (DSM-5) for substance use disorders, taking into account the increasing evidence suggesting that food addiction shares its neurobiological substrates with drug addiction (Lindgren et al., 2017). Food addiction is a complex multifactorial brain disorder resulting from the dynamic interaction among multiple gene networks and multiple environmental factors impacting brain development and function, leading to individual differences among the population (Hamer, 2002; Nestler et al., 2015). For this reason, not all individuals become addicted and extreme subpopulations can be distinguished with an addicted and non-addicted phenotype (Piazza and Deroche-Gamonet, 2013). Conversely, the precise neurobiological mechanisms underlying both phenotypes are still unclear despite the well-known common brain areas involved in addictive processes that include the basal ganglia, extended amygdala, and prefrontal cortex (Koob and Volkow, 2016; Moore et al., 2017). The current protocol improves previous studies because it has the inclusion of a short protocol for evaluating food addiction phenotype in genetically modified mice that present anticipation of food addiction development. In this protocol, the development of loss of control over food intake that characterizes addiction is revealed by measuring compulsivity, motivation, and persistence in different time-points. Compared to other operant models, this has the advantage of measuring other phenotypic traits such as impulsivity, cognitive flexibility, appetitive associative learning, and aversive conditioning. These traits are potential predictors of the development of food addiction. In this study, the main aim is to describe a replicable protocol that allows deciphering the neurobiological mechanisms involved in the resilient and vulnerable phenotypes to develop a food addiction. To address this major question, we describe a protocol with a reliable behavioral approach that can be adapted to combine a viral vector approach with chemogenetic manipulations. These findings will help to design new strategies to focus the strength in the prevention of the transition to food addiction by increasing the inhibitory control of individuals exposed to unhealthy environmental conditions.

Materials and Reagents

Materials

  1. Chocolate-flavored pellets (20 mg/pellet, 5TUL #1811223, TestDiet, Richmond, IN, USA)
  2. Microsyringe (10 μl, Model 1701 N S.Y.R., Cemented N.D.L., 26 ga, 2 in, point style 3, #80039, Hamilton company, N.V., U.S.A.)
  3. Polyethylene tubing (PE-20, #C315CT , Plastics One, U.K.)
  4. Bilateral guide cannula (26-gauge cannula cut 12 mm below pedestal, #C235GS-5/Spc, Plastics One, U.K.)
  5. Bilateral internal cannula (33-gauge internal cannula fits 12 mm C235GS-5/Spc with 3 mm projection, Plastics One, U.K.)
  6. Osmotic minipumps (flow rate of 0.25 μl/h for 28 days, Model 2004, #0000298 , Alzet, CA, U.S.A.)
  7. Scalpel ( #02-036-040 , AgnTho's, Sweden)
  8. Manual drill ( DH-1 , Plastics One, U.K.)
  9. Blunt-tipped surgical scissors ( #03-022-105 , AgnTho's, Sweden)
  10. Curved iris forceps ( #08-513-005 , AgnTho's, Sweden)
  11. Suture clips ( #08-922-125 , AgnTho's, Sweden)
  12. Surgical clips ( #22-620-007 , AgnTho's, Sweden)
  13. Suture thread (black braided silk, TB10, 3/8 TRIANG 15 mm 4/0 90 cm, #55327-50U , LorcaMarín, Spain)

Reagents
  1. Distilled water
  2. Ethanol 70%
  3. Iodine (Betadine, 500 ml, #716720 , MEDA Pharma S.A.U., Spain)
  4. Physiological saline (0.9%, 250 ml, #999790.8 , Laboratorios ERN, Spain)
  5. Glucose serum (GlucosaVet 5g/100ml, #1248 ESP , B. Braun Vet Care, Spain)
  6. Xilin night (5 g, #2919-PS-CM , Visufarma, Spain)
  7. Blastoestimulina (1%, 30g, # 719385 , Almirall, Spain)
  8. Vetflurane (Isoflurane, 250 ml, #2199-ESP , Virbac, Spain)
  9. Clozapine N-oxide (CNO, 25 mg, #BML-NS105-0025 , Enzo Life Sciences, NY) diluted in 0.9% sterile saline (5 mg/ml)
  10. Anesthesia reagents
    1. Ketamine hydrochloride (75 mg/kg of body weight, 10 ml, Ketamidor, #580393 , Richterpharma ag, Austria) dissolved in sterile 0.9% physiological saline
    2. Medetomidine hydrochloride (1 mg/kg of body weight, #570686 , Domtor; Esteve, Spain) dissolved in sterile 0.9% physiological saline
    3. Atipamezole hydrochloride (2.5 mg/kg of body weight, #570559 , Revertor; Virbac, Spain) dissolved in sterile 0.9% physiological saline
    4. Gentamicine (1 mg/kg of body weight, #999037 , Genta-Gobens; Laboratorios Normon, Spain) dissolved in sterile 0.9% physiological saline
    5. Meloxicam (2 mg/kg of body weight, Metacam; #059/02/08CVFPT , Boehringer Ingelheim, Rhein) dissolved in sterile 0.9% physiological saline
  11. Viral vectors (storage at -80 °C). Examples
    1. AAV8-hSyn-DIO-hM4D(Gi)-mCherry (1.21E + 13 gc/ml, Viral Vector Production Unit of Universitat Autònoma de Barcelona)
    2. AAV8-hSyn-DIO-mCherry (1.19E + 13 gc/ml, Viral Vector Production Unit of Universitat Autònoma de Barcelona)
    3. AAVrg pmSyn1-EBFP-Cre (6 x 1012 vg/ml, Addgene, viral prep # 51507-AAVrg )

Equipment

  1. Mouse operant self-administration chambers (Model ENV-307A-CT , Med Associates, Georgia, VT, U.S.A.)
    The operant chambers are equipped with two retractable levers (#ENV-312-2M, Med Associates), one randomly selected as the active lever and the other as the inactive. Pressing on the active lever results in a food pellet delivery paired with a stimulus-light (associated-cue, #ENV-321M, Med Associates), located above the active lever, and while pressing on the inactive lever has no consequences. A food dispenser (#ENV-303M pellet receptacle, #ENV-203M-20, modular pellet dispenser, Med Associates) equidistant between the two levers permit the delivery of food pellets when required. The floor of the chambers is a grid floor (#ENV-307A-GF, Med Associates) that serves to deliver electric foot shocks in the session of shock test and serves as a contextual cue in the session of shock-induced suppression the day after the shock test. During the rest of the self-administration sessions, a metal sheet with holes is placed above the grid floor. Thus, mice can discriminate between different contexts. A house light is placed on the ceiling of the chamber (#ENV-315M, Med Associates). The chambers are made of aluminum and acrylic and are housed in sound- and light-attenuated boxes equipped with fans to provide ventilation and white noise. (Figure 1).


    Figure 1. Diagram and images of an operant self-administration chamber. A. Diagram of the operant chamber. The operant chamber is equipped with two retractable levers (active and inactive), a stimulus-light, and a food pellet dispenser. The floor of the chamber is a grid floor that delivers electric foot shocks in the session of shock test but is covered during the rest of self-administration sessions with a metal sheet with holes. B. Image with detail of the panel containing the active lever signaled by the cue-light and the metal sheet with holes. C. General view of the Skinner Box with the operant box light, the active lever, the cue-light, the grid floor that delivers electric foot shocks, the metal sheet with holes, and the food dispenser.

  2. Stereotaxic apparatus (100-micron resolution, Model 900 , Koft instruments, C. A., U.S.A.)
  3. Standing magnifier (OPMI 1 FR, Carl Zeiss, U.S. A.)
  4. Microinfusion pump (P.H.D. 2000, #MA1 70-20xx , Harvard Apparatus, Holliston, MA, U.S.A.)
  5. Animal trimmer ( #M630 , Artero, Spain)
  6. Cold light (Leica C.L.S. 150x, Leica Microsystems, Spain)
  7. Heating pad ( #N2P 220-230 , 60W, 50Hz, Daga, Spain)
  8. Hot bead sterilizer (FST 250, #18000-45 , AgnTho's, Sweden)

Software

  1. Med-PC Software (Med Associates Inc, U.S.A.). Software that registers all the behavior in the operant self-administration chambers
  2. GraphPad Prism software (GraphPad Software, U.S.A.) to perform all graphs
  3. SPSS software (I.B.M., version 25) to perform statistical data analysis

Procedure

Male mice are housed individually in temperature (21 ± 1 °C)-and humidity (55 ± 10%) -controlled laboratory conditions maintained with food and water ad libitum. Mice are tested during the dark phase of a reverse light cycle (lights off at 8.00 a.m and on at 8.00 p.m).

  1. Self-administration session
    The beginning of each self-administration session is signaled by turning on a house light placed on the ceiling of the chamber during the first 3 s. Daily self-administration sessions maintained by chocolate-flavored pellets last 1 h in the long food addiction protocol and 2 h in the short food addiction protocol to increase the exposure of the palatable pellets on each day to ensure the development of the addiction-like phenotype. The self-administration sessions are composed of 2 pellet periods (25 min and 55 min) separated by a pellet-free period (10 min). During the pellet periods, pellets are delivered contingently after an active response paired with a stimulus light (cue light). A time-out period of 10 s is established after each pellet delivery, where the cue light is off, and no reinforcer is provided after responding on the active lever. Responses on the active lever and all the responses performed during the time-out period are recorded. During the pellet-free period, no pellet is delivered, and this period is signaled by the illumination of the entire self-administration chamber. After each session, mice are returned to their home cages.
      In the operant conditioning sessions, mice are under a fixed ratio 1 (FR1) schedule of reinforcement (1 lever-press results in one pellet delivery) followed by an increased F.R. to 5 (FR5) (5 lever-presses results in 1 pellet delivery) for the rest of the sessions. As previously described (Martín-García et al., 2011), the criteria for the achievement of the operant responding are acquired when all of the following conditions are met: (1) mice maintained a stable responding with less than 20% deviation from the mean of the total number of reinforcers earned in 3 consecutive sessions (80% of stability); (2) at least 75% responding on the active lever; and (3) a minimum of 5 reinforcers per session.

  2. Measurement of the 3 food addiction-like criteria
    Three behavioral tests are used to evaluate the food addiction-like criteria as described (Mancino et al., 2015; Domingo-Rodriguez et al., 2020) and adapted from cocaine addiction-like in rats (Deroche-Gamonet et al., 2004). These 3 criteria summarize the hallmarks of addiction based on DSM-IV (Piazza and Deroche-Gamonet, 2013), specified in DSM-5, and now included in the food addiction diagnosis through the YFAS 2.0 (Gearhardt et al., 2016).
    1. Persistence to response (Figure 2A): Non-reinforced active responses during the pellet free period (10 min), when the box is illuminated and signaling the unavailability of pellet delivery, are measured as persistence of food-seeking behavior. On the 3 consecutive days before the progressive ratio, mice are scored.
    2. Motivation (Figure 2B): The progressive ratio schedule of reinforcement is used to evaluate the motivation for chocolate-flavored pellets. The response required to earn one single pellet escalates according to the following series: 1, 5, 12, 21, 33, 51, 75, 90, 120, 155, 180, 225, 260, 300, 350, 410, 465, 540, 630, 730, 850, 1,000, 1,200, 1,500, 1,800, 2,100, 2,400, 2,700, 3,000, 3,400, 3,800, 4,200, 4,600, 5,000, and 5,500. The maximal number of responses that the animal performs to obtain one pellet is the last event completed, referred to as the breaking point. The maximum duration of the progressive ratio session is 5 h or until mice do not respond on any lever within 1 h.
    3. Compulsivity (Figure 2C): Total number of shocks in the session of shock test (50 min), when each pellet delivered is associated with punishment, are used to evaluate compulsivity-like behavior, previously described as resistance to punishment (Deroche-Gamonet et al., 2004; Mancino et al., 2015). Mice are placed in a self-administration chamber without the metal sheet with holes and, consequently, with the grid floor exposed (contextual cue). In this shock-session, mice are under an FR5 schedule of reinforcement during 50 min with 2 schedule changes: at the 4th active lever-response mice receive only an electric foot-shock (0.18 mA, 2 s) without pellet delivery, and at the 5th active lever-response, mice receive another electric foot-shock with a chocolate-flavored pellet paired with the cue light. The schedule is reinitiated after 10 s pellet delivery (time-out period) and after the 4th response if mice do not perform the 5th response within 1 min.


      Figure 2. The 3 addiction-like criteria. A. Persistence to response. Non-reinforced active responses during the pellet free period (10 min), when the box is illuminated and signaling the unavailability of pellet delivery, are used to evaluate the persistence of food-seeking. B. Motivation. The progressive ratio test is used to evaluate the motivation for chocolate-flavored pellets. The responses to earn a single pellet increase exponentially. The maximal number of responses that the animal performs to obtain one pellet is the last event completed, referred to as the breaking point. C. Compulsivity. The total number of shocks in the session of shock test, when each pellet delivered is associated with punishment, is used to evaluate compulsivity-like behavior.

    4. Attribution of the 3 addiction-like criteria (Figure 3): After performing the 3 behavioral tests to measure the food addiction-like behavior, mice are categorized in vulnerable or resilient animals at the early period and addicted or non-addicted animals at the late period, depending on the number of positive criteria that they have achieved at the early or late period respectively. An animal is considered positive for an addiction-like criterion when the score of the specific behavioral test is equal or above the 75th percentile of the normal distribution of the chocolate control group. Mice that achieve 2 or 3 addiction-like criteria are considered vulnerable, or addicted animals and mice that achieve 0 or 1 addiction-like criteria are considered resilient or non-addicted animals.
    Note: The example of graphs presented in this section are the data obtained with CBL57/6N mice. Data derived from Domingo-Rodriguez et al., 2020.


    Figure 3. Attribution of the 3 addiction-like criteria Mice perform 3 behavioral tests to measure the food addiction-like behavior and obtain an individual score for each criterion. The percentile 75th of the normal distribution of the chocolate control group in each criterion (dashed horizontal line) is established as a threshold to consider an animal positive for this addiction-like criterion when its individual score is equal or above the 75th percentile. Mice that achieve 2 or 3 addiction-like criteria are considered addicted animals, and mice that achieve 0 or 1 addiction-like criteria are considered non-addicted animals. Four mice (A-D) are indicated in the figure as an example. Mouse A presents the values of each criterion below the threshold achieving 0 criteria and is classified as a non-addicted animal. Mouse B displays a score in persistence to the response above the threshold and below in motivation and compulsivity, achieving 1 criterion and is classified as a non-addicted animal. Mouse C shows a score in persistence to response and compulsivity above the threshold and below in motivation achieving 2 criteria and is categorized as an addicted animal. Mouse D shows a score of each criterion above the 75th percentile achieving 3 criteria and is classified as an addicted animal. Data are expressed as individual values and median with interquartile range. White circles: mice with 0 criteria. Green circles: mice with 1 criterion. Blue circles: Mice with 2 criteria. Red circles: mice with 3 criteria. Data derived from Domingo-Rodriguez et al., 2020.


  3. Measurement of 4 phenotypic traits considered as factors of vulnerability to addiction-like behavior
    1. Impulsivity (Figure 4A): Non-reinforced active responses during the time-out periods (10 s) after each pellet delivery are measured as impulsivity-like behavior indicating the inability to stop a response once it is initiated (Logan et al., 1997; Koob and Volkow, 2010). The 3 consecutive days before the progressive ratio test are considered for this criterion.
    2. Cognitive flexibility (Figure 4B): Measured with a reversal test that indicates the ability to change responding to stimuli that have previously predicted the availability of reward (Schoenbaum et al., 2011). The reversal test is a standard training self-administration session, but the active and the inactive levers are reversed.
    3. Appetitive associative learning (Figure 4C): Measured with the cue-induced food seeking test. The cue-induced food-seeking test lasts 90 min and is divided into two periods: 60 min + 30 min. In the first 60 min period, all lever-presses are not reinforced (active and inactive lever-presses have no scheduled consequences). In the subsequent 30 min, the white cue light previously associated with pellet delivery during a normal self-administration session is illuminated contingently for 30 min according to an FR5. To signal the change in the schedule, the cue light is presented twice non-contingently and for 4 s.
    4. Aversive associative learning (Figure 4D): Measured with the shock-induced suppression test. In the shock-induced suppression test, mice are placed in the self-administration chamber for 50 min with the same grid floor used during the shock-test. However, during this FR5 self-administration session, pressing the active lever has no consequences, no shock, no chocolate-flavored pellets, and no cue-light. Non-reinforced active responses during this shock-induced suppression test are measured for the aversive associative learning.


    Figure 4. The 4 phenotypic traits considered as factors of vulnerability to addiction-like behavior. A. Impulsivity measured by the non-reinforced active responses not paired with a stimulus light during the time-out periods (10 s) after each pellet delivery. B. Cognitive flexibility measured by the reversal test. The reversal test is an FR5 self-administration session, but the active and the inactive levers are reversed compared to the previous self-administration session. C. Appetitive associative learning measured by the cue-induced food-seeking test. The cue-induced food-seeking test is a self-administration session that longs 90 min and is divided into two periods: 60 min + 30 min. In the first 60 min period, all lever-presses are not reinforced (active and inactive lever-presses have no scheduled consequences). In the subsequent 30 min, the white cue light, associated with pellet delivery during a normal self-administration session, is illuminated contingently for 30 min according to an FR5. D. Aversive associative learning measured by the shock-induced suppression test.


  4. Experimental design
    Long protocol for food addiction in mice (Figure 5): In the long food protocol, mice are trained under FR1 schedule of reinforcement during 6 sessions, followed by 112 sessions of FR5 to self-administer chocolate-flavored pellets. During FR5 sessions, the 3 addiction-like criteria (1) persistence to response (2), motivation (3), and compulsivity and 4 phenotypic traits considered as factors of vulnerability to addiction (1) impulsivity, (2) cognitive flexibility, (3) appetitive associative learning and (4) aversive associative learning, are evaluated at 2 different time points in each mouse. The 1st time point is the early period (sessions 1-18 of FR5), and the 2nd time point is the late period (sessions 95-112 of FR5). Depending on the positive criteria that mice have achieved in the early period, animals are categorized in resilient (0-1 criteria) or vulnerable animals (2-3 criteria), and in the late period, mice are categorized in non-addicted (0-1 criteria) or addicted animals (2-3 criteria).


    Figure 5. Timeline of the experimental sequence of the long food addiction mouse model. Mice are trained for chocolate-flavored pellets under an FR1 schedule of reinforcement on 1 h daily sessions for 6 days, followed by 112 days on an FR5. In the FR5, 2-time points are considered, early and late period, to measure the 3 addiction-like criteria (persistence to response, motivation, and compulsivity). Depending on the positive criteria that mice have achieved in the early period, animals are categorized in resilient (0-1 criteria) or vulnerable animals (2-3 criteria), and in the late period, mice are categorized in non-addicted (0-1 criteria) or addicted animals (2-3 criteria). In both early and late periods, 4 phenotypic traits as factors of vulnerability to addiction (impulsivity, cognitive flexibility, appetitive associative learning, and aversive associative learning) are also evaluated.


  5. Experimental design
    Short protocol for food addiction in mice (Figure 6): In the short protocol, mice follow the same behavioral procedure described for the early period in the long food addiction protocol with some variations due to the surgical viral vector injection. The short protocol lasts 9 weeks. In the 1st week, mice are trained to acquire the operant conditioning maintained by chocolate-flavored pellets under FR1 (2 sessions) and FR5 (2 sessions) schedules of reinforcement. In the 2nd week, the viral vector of interest is injected in mice by stereotaxic surgery.
    Examples of viral vectors strategies:
    1. Chemogenetic approach (DREADD approach): It refers to the injection of Cre-dependent AAV carrying a DREADD in a determined brain region of genetically modified mice expressing the Cre-recombinase in a specific cell-type.
    2. Combined chemogenetic and a retrograde AAV approach (retro-DREADD approach): It refers to the combinatorial injection of Cre-dependent AAV carrying a DREADD in a determined brain region and an AAV retrograde expressing the Cre-recombinase in its projecting brain area to target a specific network in wild type mice.
      After bilateral intracranial injection/s of the viral vector/s, the expression of the viral vector/s is allowed during the period of 4 weeks (2nd, 3rd, 4th, and the 5th week). At the beginning of this period (3rd week), mice are under FR5 (4 sessions) to recover the basal levels of operant responding. At the end of these 4 weeks (5th week), an osmotic minipump filled with CNO or saline is subcutaneously implanted in the back of each mouse. Subsequently, during the 6th, 7th, 8th, and 9th weeks, when it is a chronically CNO-induced activation of the expressed DREADD receptors, mice are under FR5 scheduled sessions followed by the measurement of the 3 addiction-like criteria. Finally, depending on the positive criteria that mice have achieved, animals are categorized in non-addicted (0-1 criteria) or addicted animals (2-3 criteria).


      Figure 6. Timeline of the experimental sequence of the short food addiction mouse model. In the 1st week, mice are trained to acquire the operant conditioning maintained by chocolate-flavored pellets under FR1 (2 sessions) and FR5 (3 sessions) schedule of reinforcement followed by the surgery for injecting the viral vector of interest (2nd week). After surgery, the expression of the viral vector is allowed for a period of 4 weeks (2nd, 3rd, 4th, and the 5thweek). At the beginning of this period (3rd week), mice are nder FR5 (4 sessions) to recover the basal levels of operant responding, and at the end of this period (5th week), an osmotic minipump filled with CNO is implanted. During the 6th, 7th, 8th, and 9th weeks, when it is a chronic inhibition of the CNO-induced activation of the expressed DREADD receptors, mice are under FR5 scheduled sessions followed by the measurement of the 3 addiction-like criteria.

    Step by step protocol
    1. Long food addiction mouse protocol
      1. Sessions 1-6 (FR1): During these sessions, operant conditioning under the FR1 schedule of reinforcement was applied.
      2. Sessions 1-112 (FR5): During these sessions, operant conditioning under the FR5 schedule of reinforcement was applied.
        1. Sessions 1-18 (FR5): These sessions of FR5 constituted the early period of the long food addiction mouse protocol.
          1. Sessions 3-5: FR5 sessions to evaluate the criterion of persistence to response and the phenotypic trait of impulsivity were applied.
          2. 1)
            The non-reinforced active responses during the pellet free period (10 min) are used to evaluate the persistence to response criteria.
            2)
            The non-reinforced active responses during the time-out periods (10 s) after each pellet delivery are used to evaluate the impulsivity phenotypic trait.
          3. Session 6: Progressive ratio test to evaluate the motivation criteria.
          4. Session 10: Shock test to evaluate the compulsivity criteria.
          5. Session 11: Shock-induced suppression test to evaluate the aversive associative learning phenotypic trait.
          6. Session 15: Cue-induced food-seeking test to evaluate appetitive associative learning phenotypic trait.
          7. Session 18: Reversal test to evaluate cognitive flexibility phenotypic trait.
        2. Categorize mice in vulnerable and resilient animals depending on the number of positive addiction-like criteria achieved at the early period.
        3. Sessions 18-95 (FR5): FR5 sessions.
        4. Sessions 95-112 (FR5): Theses sessions constitute the late period.
          1. Sessions 97-99: Normal FR5 sessions to evaluate the criterion of persistence to response and the phenotypic trait of impulsivity.
          2. 1)
            The non-reinforced active responses during the pellet free period (10 min) are used to evaluate the persistence to response criteria.
            2)
            The non-reinforced active responses during the time-out periods (10 s) after each pellet delivery are used to evaluate the impulsivity phenotypic trait.
          3. Session 100: Progressive ratio test to evaluate the motivation criteria.
          4. Session 104: Shock test to evaluate the compulsivity criteria.
          5. Session 105: Shock-induced suppression test to evaluate the aversive associative learning phenotypic trait.
          6. Session 109: Cue-induced food-seeking test to evaluate appetitive associative learning phenotypic trait.
          7. Session 112: Reversal test to evaluate cognitive flexibility phenotypic trait.
        5. Categorize mice in addicted and non-addicted animals depending on the number of positive addiction-like criteria achieved in the late period. Using this protocol in C57BL/6N, we obtained 25% of addicted mice.

    2. Short food addiction mouse protocol
      1. Week 1: Learning of the operant responding to obtain chocolate-flavored pellets.
        1. Session 1-2: Operant conditioning under the FR1 schedule of reinforcement.
        2. Session 3-4: Operant conditioning under the FR5 schedule of reinforcement.
      2. Week 2: Injection of a viral vector by stereotaxic surgery.
      3. Stereotaxic surgery protocol (Figure 7)
        Note: Sterilize the surgical material with a hot bead sterilizer before each animal surgery.
        1. Injection cannulas circuit preparation.
          1. Remove the plunger of the microsyringe (10 µl).
          2. Attach the polyethylene tubing (50 cm) to the microsyringe.
          3. Inject distilled water through the tubing, using a syringe, filling the tubing, and the microsyringe. Check that the water comes out through the back of the microsyringe.
          4. Insert half of the plunger into the microsyringe.
          5. Connect one of the sides of the bilateral internal cannula (33 gauge) to the tubing. Check that the circuit is well done by pressing the microsyringe plunger and observing how the water flows out of the cannula without leakage.
          6. Repeat these steps with a second microsyringe, which must be attached to the other side of the same internal cannula as the first microsyringe.
          7. Place the microsyringes in the microinfusion pump.
          8. Aspirate 1 µl of air to form a bubble inside the tubing to monitor the microinjections.
          9. Aspirate 4 µl of the viral vector (previously defrosted with ice).
        2. Stereotaxic apparatus preparation.
          1. Place and fix the bilateral guide cannula in the metal grip of the stereotaxic holder.
          2. Insert the bilateral internal cannula attached to the tubing into the bilateral guide cannula. The bilateral guide cannula is used to keep the internal cannula straight and well fitted into the holder and to find the Bregma coordinates before the AAV is loaded. Note that only the internal cannula filled with the viral vector is the one that penetrates the brain.
        3. Mouse preparation.
          1. Anesthetize the mouse with ketamine hydrochloride and medetomidine hydrochloride mixed and dissolved in sterile 0.9% physiological saline. Administer intraperitoneally (75 mg/kg and 1 mg/kg of body weight, respectively).
          2. Shave the mouse's head.
          3. Place mouse in the stereotaxic apparatus. Check that the head is fixed using the ears bars and the nose clamp.
          4. Place a heating pad below the mouse to maintain proper body temperature during the surgery.
        4. Stereotaxic surgery.
          1. Apply iodine to the shaved head area.
          2. Apply xilin night on eyes to avoid keratitis.

            Note: The following steps must be done using a standing magnifier.

          3. Perform a vertical cut in the middle of the head with a scalpel.
          4.  Scratch the skull carefully to visualize bregma accurately.
          5. Place the cannulas above the bregma and annotate bregma coordinates.
          6. Calculate medial-lateral, dorsal-ventral, and anteroposterior coordinates of your target area where the injection has to be performed.
          7. Move the cannulas to the specific injection point by adjusting the stereotaxic apparatus using the medial-lateral, dorsal-ventral, and anterior-posterior readings.
          8. Mark the injection point on the skull using a pencil.
          9. Following the pencil mark, perform the holes into the skull using a manual drill.
          10. Clean the injection area with physiological saline.
          11. Insert the bilateral cannula into the holes and readjust the dorsal-ventral coordinate.
          12. Place precisely the bilateral cannulas following the calculated coordinates. 
          13. Mark the two sides of the air bubble on the tubing to monitor the injection.
          14. Perform the viral vector injection.
          15. 1)
            Microinfusion pump parameters (example for prelimbic (P.L.) and nucleus accumbens core (NAc core) areas)

            • Target volume: PL 0.2 µl ; NAc core 0.4 µl
            • Infusion rate: PL 0.05 µl/min; NAc core 0.1 µl/min
            • Infusion time: 4 min

            2)
            Leave the cannula in place after infusion for 10 min to prevent reflux.
            3)
            Withdrawn the cannula slowly for 10 min.
          16. Clean the injection area with physiological saline.
          17. Sew the incision with suture thread.
          18. Apply blastoestimulina on the sutured incision to promote true healing.
          19.  Inject subcutaneously atipamezole hydrochloride (2.5 mg/kg of body weight) dissolved in sterile 0.9% physiological saline to reverse the anesthesia.
          20. Inject intraperitoneally gentamicin (1 mg/kg of body weight) dissolved in sterile 0.9% physiological saline.
          21. Inject meloxicam subcutaneously (2 mg/kg of body weight) dissolved in sterile 0.9% physiological saline.
          22. Inject glucose serum (0.8 ml) subcutaneously.
          23. Place the animal on a heating pad until the animal is awake.
          24. During the following 3 postsurgery days, check the mouse and inject 0.8 ml of glucose serum every day.
        5. Cleaning.
          1. Clean the tubing and the cannulas with ethanol, followed by distilled water and air.
          2. Clean all the surgical material with soap and water.
            After the bilateral intracranial injection, 4 weeks (weeks 2, 3, 4, and 5 in this protocol) are needed for the proper expression of the viral vector.


          Figure 7. Stereotaxic surgery. Mice are anesthetized and placed into a stereotaxic apparatus for receiving the viral vector intracranial injections. All the injections are made through a bilateral injection cannula connected to a polyethylene. The displacement air bubble inside the length of the polyethylene tubing that connected the syringe to the injection needle is used to monitor the microinjections. The viral vector is injected at a constant rate by using a microinfusion pump. After infusion, the injection cannula is left in place for an additional period of 10 min to allow the fluid to diffuse and to prevent reflux, and then it was slowly withdrawn during 10 additional min.
          Note: Frequently check the correct displacement of the air bubble inside the polyethylene tube to prevent errors of injection due to obturation of the cannula or fugues in the tube.

      4. Week 3: Operant conditioning after surgery to recover the basal levels of responding.
        Sessions 5-9: Operant conditioning under the FR5 schedule of reinforcement.
      5. Week 4: Operant conditioning.
      6. Week 5: At the end of the week implant, an osmotic minipump filled with CNO or saline.
      7. Osmotic minipump implantation protocol (Figure 8)
        1. Anesthetize the mouse with isoflurane and fix its front and hind legs with tape.
        2. Shave mouse by the lower back.
        3. Clean the shaved lower back with ethanol.
        4. Make a horizontal cut (1.5 cm), taking as a reference the beginning of the hind legs.
        5. Separate the skin from the back muscle introducing blunt-tipped scissors through the cut.
        6. Introduce the osmotic minipump (ALZET 2004) previously filled with CNO (diluted in 0.9% sterile saline: 5 mg/ml) or saline subcutaneously in the middle of the back. The pump cap in the direction of the animal's head.
        7. Close the cut with surgical clips (around 4).
        8. Apply iodine to the area.



        Figure 8. Schematic representation of the osmotic minipump implantation process. A. Anesthetize the mouse with isoflurane and shave it by the lower back. Clean the shaved area with ethanol. B. Make a horizontal cut (1.5 cm), taking as a reference the beginning of the hind legs. C. Separate the skin from the back muscle introducing blunt-tipped scissors through the cut. D. Introduce the osmotic minipump previously filled with CNO or saline subcutaneously in the middle of the back. The pump cap in the direction of the animal's head. Close the cut with surgical clips and apply iodine to the area. E. Side view showing the animal awake with the subcutaneously implanted minipump.
        Note: Check the correct placement of the minipump the following days after surgery. If necessary, do a smooth massage in the placement site of the minipump to relocate and prevent skin wounds.

      8. Week 6: Operant conditioning during the expression of the viral vector activated by CNO.
        Sessions 10-14: Operant conditioning under the FR5 schedule of reinforcement.
      9. Week 7: Operant conditioning during the expression of the viral vector chronically activated by CNO.
        Sessions 15-20: Operant conditioning under the FR5 schedule of reinforcement.
      10. Week 8: Operant conditioning during the expression of the viral vector chronically activated by CNO.
        1. Sessions 21: Operant conditioning under the FR5 schedule of reinforcement.
        2. Session 22-24: Normal FR5 sessions to evaluate persistence to response and impulsivity.
          1. The non-reinforced active responses during the pellet free period (10 min) are used to evaluate the persistence to response criteria.
          2. The non-reinforced active responses during the time-out periods (10 s) after each pellet delivery are measured as impulsivity-like behavior.
        3. Session 25: Progressive ratio test to evaluate the motivation criteria.
      11. Week 9: Operant conditioning during the expression of the viral vector chronically activated by CNO.
        1. Sessions 26-28: Operant conditioning under FR5 schedule of reinforcement.
        2. Session 29: Shock test to evaluate the compulsivity criteria.
        3. Session 30: Shock-induced suppression test to evaluate the aversive associative learning.
      12. Categorize mice in addicted and non-addicted animals depending on the number of positive addiction-like criteria achieved. Using this protocol in C57BL/6J mice with an inhibition of the PL-NAc core pathway by chemogenetic approach, we obtained 50% of addicted mice.
      13. Verify the viral expression.
        1. Perfuse animals using intracardiac perfusion with 4% paraformaldehyde (P.F.A.) in 0.1 M Na2HPO4/0.1 M NaH2PO4 buffer (P.B.), pH 7.5, delivered with a peristaltic pump at 30 ml per min for 2 min. Animals are previously profoundly anesthetized by intraperitoneal injection (0.2 ml/10 g of body weight) of a mixture of ketamine/medetomidine.
        2. Extract the brains and post-fixed with 4% P.F.A. for 24 h and transfer them to a solution of 30% sucrose at 4 °C.
        3. Make coronal sections (30 μm) using a freezing microtome and store them in a 5% sucrose solution at 4 °C until immunofluorescence study.
        4. Perform an immunofluorescence study using the specific antibodies for your viral vector fluorescent reporter used.
        5. Visualize the stained sections of the brain with a confocal microscope to evaluate histological verification of correct injection placement.
        6. Make representative immunofluorescence pictures (Figure 9).


        7. Figure 9. Representative immunofluorescence picture showing Cre-dependent hM4Di-mCherry detected at P.L. injection site (left) and Cre recombinase at NAc core (right). Data derived from Domingo-Rodriguez et al., 2020.


    Data analysis

    1. Exclusion criteria
      1. Animals that responded < 25% of all FR5 sessions and did not achieve the acquisition criteria were excluded from the remaining experimental sequence.
      2. Animals with a viral vector expression outside the brain regions of interest are excluded from the experiment.

    2. Graphs and statistical analysis
    1. Number of reinforcers
      1. Graphs: Mean number of reinforcers obtained in each session during the entire food addiction protocol per group (Figure 10). Mean ± S.E.M represents data.
      2. Statistical analysis: ANOVA analyzes the number of reinforcers with repeated measures for each step of the protocol separately (FR1, FR5) to test the evolution over time.


        Figure 10. Chemogenetic inhibition of PL-NAc core projection leads to compulsive behavior towards highly palatable food. Number of reinforcers during operant training sessions maintained by chocolate-flavored pellets (mean ± S.E.M). In white saline-treated mice and red for CNO-treated mice (n = 12 for saline-treated mice and n = 22 for CNO-treated mice).

      3. Behavioral tests of the 3 addiction-like criteria (Persistence to response, motivation, and compulsivity)
        1. Graphs: The data of the 3 criteria are expressed as individual values with the median and the interquartile range (Figure 11).
          1. Persistence to response: Mean of the total number of non-reinforced active responses during 3 consecutive daily 10-min of pellet free period.
          2. Motivation: Breaking point achieved in 5 h of the progressive ratio test.
          3. Compulsivity: Number of shocks that mice received in 50 min in the shock test in which each pellet delivery is associated with a foot-shock.
          4. The graphs of the 3 addiction-like criteria at the late period present a dashed horizontal line indicating the 75th percentile of the distribution of the control group used as the threshold to consider a mouse positive for 1 criterion.


            Figure 11. Behavioral tests of the three addiction-like criteria showing increased compulsivity in CNO-treated mice with an inhibition of the PL-NAc core pathway (individual values with the median and the interquartile range, U Mann-Whitney, **P < 0.01). The dashed horizontal line indicates the 75th percentile of the distribution of mice treated with saline. Addicted mice in gray filled circles for saline-treated mice and red for CNO-treated mice (n=12 for saline-treated mice and n = 22 for CNO-treated mice).

          5. Statistical analysis
            1. Analyze the distribution of the sample by the Kolmogorov-Smirnov normality test.
            2. Comparisons between the two groups are analyzed by U Mann-Whitney if the sample does not follow a normal distribution (significance value in the Kolmogorov-Smirnov normality test).
            3. Comparisons between groups are analyzed by Student’s t-test if the sample follows a normal distribution (no significant value in the Kolmogorov-Smirnov normality test).
        2. Categorization of mice in addicted and non-addicted animals
          1. Graphs: Parts of holes graphs represent the percentage of addicted and non-addicted animals in each group (Figure 12).
          2. Statistical analysis: The percentage of addicted mice compare with the non-addicted ones are analyzed by a Chi-square test, in which it is compared the observed frequencies with the frequencies obtained in the control group.


            Figure 12. Increased percentage of CNO-treated with an inhibition of the PL-NAc core pathway mice classified as food addicted animals (chi-square, ***P < 0.001, n = 12 for saline-treated mice and n = 22 for CNO-treated mice).

          3. Correlations between individual values of each addiction-like criteria and the final number of positive criteria achieved. Correlations are analyzed by Pearson's correlation coefficient.
          4. Behavioral tests of the 4 phenotypic traits as factors of vulnerability to addiction-like behavior (Impulsivity, cognitive flexibility, appetitive associative learning, and aversive associative learning):
            1. Graphs: The data of the 4 phenotypic traits are expressed as individual values with the median and the interquartile range.
              1. Impulsivity: Mean number of non-reinforced active responses in 50 min during 3 consecutive daily time-out (10 s) after each pellet delivery.
              2. Cognitive flexibility: Number of active and inactive responses in 60 min of the reversal test where active and inactive levers were reversed compared with preceding basal session.
              3. Appetitive associative learning:
                1)
                Active responses during 60 min period of the cue-induced food-seeking test, during which lever-presses were not reinforced, followed by active responses 30 min period during which active lever-presses (FR5) were associated with the cue-light without pellet delivery (mean ± S.E.M).
                2)
                Increase of active responses after the presentation of the cue-light.
              4. Aversive associative learning: Number of non-reinforced active responses in 50 min of the shock-induced suppression test.
            2. Statistical analysis
              i. Analyze the distribution of the sample by the Kolmogorov-Smirnov normality test.
              ii. Comparisons between the two groups are analyzed by U Mann-Whitney if the sample does not follow a normal distribution (significance value in the Kolmogorov-Smirnov normality test).
              iii. Comparisons between groups are analyzed by Student’s t-test if the sample follows a normal distribution (no significant value in the Kolmogorov-Smirnov normality test).
          5. A P-value < 0.05 is used to determine statistical significance.

          Expected outcome
          Note: The example of graphs presented in this section are the data obtained comparing mice with a chemogenetic inhibition of PL-NAc core pathway and mice with no inhibition of this network. All this data is published in the manuscript Domingo-Rodriguez et al., 2020.

      Notes

      Bodyweight and food intake are measured once a week during the entire short and long food addiction mouse protocols. These measurements are especially crucial in the short food addiction mouse protocol in which it is used osmotic minipumps filled with CNO. We demonstrated that in our conditions, no side effects of CNO are revealed on body weight, food intake, and neither in locomotor activity (Domingo-Rodriguez et al., 2020).

      Acknowledgments

      We thank E. Senabre, S. Kummer for their critics and technical support. This work was supported by the Spanish Ministerio de Economía y Competitividad-MINECO (#SAF2017-84060-R-AEI/FEDER-UE), the Spanish Instituto de Salud Carlos III, RETICS-RTA (#RD12/0028/0023), the Generalitat de Catalunya, AGAUR (#2017 SGR-669), ICREA-Acadèmia (#2015) and the Spanish Ministerio de Sanidad, Servicios Sociales e Igualdad, Plan Nacional Sobre Drogas (#PNSD-2017I068) to R.M., Fundació La Marató-TV3 (#2016/20-30) and Plan Nacional Sobre Drogas of the Spanish Ministry of Health (#PNSD-2019I006) to E.M-G. The methodology described was previously used in Domingo-Rodriguez et al., 2020. Figures with drawings are created with BioRender.com.

      Competing interests

      The authors have no conflicts of interest.

      Ethics

      All experimental protocols were performed in accordance with the guidelines of the European Communities Council Directive 2010/63/EU and approved by the local ethical committee (Comitè Ètic d'Experimentació Animal-Parc de Recerca Biomèdica de Barcelona, CEEA-PRBB, agreement N 9687).

      References

      1. Deroche-Gamonet, V., Belin, D. and Piazza, P. V. (2004). Evidence for addiction-like behavior in the rat. Science 305(5686): 1014-1017.
      2. Domingo-Rodriguez, L., Ruiz de Azua, I., Dominguez, E., Senabre, E., Serra, I., Kummer, S., Navandar, M., Baddenhausen, S., Hofmann, C., Andero, R., Gerber, S., Navarrete, M., Dierssen, M., Lutz, B., Martín-García, E. And Maldonado, R. (2020). A specific prelimbic-nucleus accumbens pathway controls resilience versus vulnerability to food addiction. Nat Commun 11(1): 782.
      3. Gearhardt, A. N., Corbin, W. R. and Brownell, K. D. (2016). Development of the Yale Food Addiction Scale Version 2.0. Psycho Addict Behav 30(1): 113-121.
      4. Hamer, D. (2002). Genetics. Rethinking behavior genetics. Science 298(5591): 71-72.
      5. Koob, G. F. and Volkow, N. D. (2010). Neurocircuitry of addiction. Neuropsychopharmacology 35(1): 217-238.
      6. Koob, G. F. and Volkow, N. D. (2016). Neurobiology of addiction: a neurocircuitry analysis. Lancet Psychiatry 3(8): 760-773.
      7. Lindgren, E., Gray, K., Miller, G., Tyler, R., Wiers, C. E., Volkow, N. D. and Wang, G. J. (2018). Food addiction: A common neurobiological mechanism with drug abuse. Front Biosci (Landmark Ed) 23: 811-836.
      8. Logan, G. D., Schachar, R. J. and Tannock, R. (1997). Impulsivity and Inhibitory Control. Psychol Sci 8(1): 60-64.
      9. Mancino, S., Burokas, A., Gutierrez-Cuesta, J., Gutierrez-Martos, M., Martin-Garcia, E., Pucci, M., Falconi, A., D'Addario, C., Maccarrone, M. and Maldonado, R. (2015). Epigenetic and Proteomic Expression Changes Promoted by Eating Addictive-Like Behavior. Neuropsychopharmacology 40(12): 2788-2800.
      10. Martin-Garcia, E., Burokas, A., Kostrzewa, E., Gieryk, A., Korostynski, M., Ziolkowska, B., Przewlocka, B., Przewlocki, R. and Maldonado, R. (2011). New operant model of reinstatement of food-seeking behavior in mice. Psychopharmacology (Berl) 215(1): 49-70.
      11. Moore, C. F., Sabino, V., Koob, G. F. and Cottone, P. (2017). Pathological Overeating: Emerging Evidence for a Compulsivity Construct. Neuropsychopharmacology 42(7): 1375-1389.
      12. Nestler, E. J., Pena, C. J., Kundakovic, M., Mitchell, A. and Akbarian, S. (2016). Epigenetic Basis of Mental Illness. Neuroscientist 22(5): 447-463.
      13. Piazza, P. V. and Deroche-Gamonet, V. (2013). A multistep general theory of transition to addiction. Psychopharmacology (Berl) 229(3): 387-413.
      14. Pursey, K. M., Stanwell, P., Gearhardt, A. N., Collins, C. E. and Burrows, T. L. (2014). The prevalence of food addiction as assessed by the Yale Food Addiction Scale: a systematic review. Nutrients 6(10): 4552-4590.
      15. Schoenbaum, G., Roesch, M. R., Stalnaker, T. A. and Takahashi, Y. K. (2011). Orbitofrontal Cortex and Outcome Expectancies: Optimizing Behavior and Sensory Perception. In: Gottfried, J. A. (Ed.). Neurobiology of Sensation and Reward. Boca Raton (FL): CRC Press/Taylor & Francis.

简介

[摘要]对食物成瘾的研究包括3个标志,包括对反应的坚持 没有结果,就很难获得可口食物的动力,失去对食物摄入的抑制性控制会导致成瘾者的强迫行为。这种疾病的复杂的多因素性质和未知的神经生物学机制相关性解释了缺乏有效的治疗方法。我们的操作员条件模型可以解释为什么有些人容易受到伤害并发展成瘾,而另一些人却坚强不屈。由于它基于《精神疾病诊断和统计手册》第5版(DSM-5)和耶鲁大学食物成瘾量表(YFAS 2.0),因此是一种翻译方法。该模型可以通过将其分为以下两种情况来评估成瘾标准:在两个时间点上的早期和晚期:1)在没有食物的时期内对反应的持续性; 2)以渐进的比例寻找食物的动机;以及3)当奖励与诸如电击脚之类的惩罚相关联时的强迫性。该模型的优势在于,它允许我们测量4个表型性状,这些性状是与成瘾易感性相关的诱发因素。同样,可以用转基因小鼠评估长期食物成瘾小鼠模型。重要的是,该协议的新颖性在于使该食物成瘾模型适应于一种简短协议,以通过使用能够促进这种成瘾行为迅速发展的化学生成方法来评估针对特定大脑回路的基因操作。这些适应导致了短食物成瘾小鼠方案,其中小鼠遵循长食物成瘾方案中早期的相同行为程序,但由于手术病毒载体注射而有所不同。据我们所知,小鼠中没有范式可以让我们研究这种健壮的行为方法的组合,这种方法可以在大脑回路层面揭示食物成瘾的神经生物学。我们可以研究使用此协议,如果修改特定大脑网络的兴奋性赋予韧性或易感性。理解这些神经生物学机制有望帮助找到新颖有效的干预措施来对抗食物成瘾。

[背景]在过去的几年中,由于全球成瘾率的提高(19.9%),食物成瘾引起了人们的关注,目前对个人和社会造成了沉重的负担,而没有任何有效的治疗方法(Pursey等,2014)。当前的诊断由最近验证的工具耶鲁食品成瘾量表2.0(YFAS 2.0)执行。该仪器基于第五版《精神疾病统计手册》(DSM-5)中针对物质使用障碍的标准,同时考虑到越来越多的证据表明食物成瘾与药物成瘾具有相同的神经生物学底物(Lindgren等人。,2017) 。食物成瘾是由多种基因网络和影响大脑发育和功能的多种环境因素之间的动态相互作用导致的复杂的多因素脑部疾病,导致人群之间的个体差异(Hamer,2002; Nestler等,2015)。由于这个原因,并不是所有的人都会上瘾,极端的亚群可以通过上瘾和不上瘾的表型来区分(Piazza和Deroche-Gamonet,2013)。相反,尽管参与成瘾过程的众所周知的常见大脑区域包括基底神经节,延伸杏仁核和前额叶皮层,但仍不清楚这两种表型的确切神经生物学机制(Koob和Volkow,2016年; Moore等人,201 7 )。目前的方案改进了先前的研究,因为它包含了一个简短的方案,用于评估对食物成瘾发展有预期的转基因小鼠的食物成瘾表型。在该协议中,通过测量在不同时间点的强迫性,动机和持续性,揭示了表征成瘾的对食物摄入失去控制的发展。与其他操作模型相比,这具有测量其他表型特征的优势,例如冲动性,认知灵活性,竞争性联想学习和厌恶条件。这些特征是食物成瘾发展的潜在预测因子。在这项研究中,主要目的是描述一种可复制的方案,该方案允许破译涉及有弹性和脆弱表型的神经生物学机制以发展食物成瘾。为了解决这个主要问题,我们描述了一种具有可靠行为方法的协议,该协议可以适用于将病毒载体方法与化学发生学操作相结合。这些发现将有助于设计新的策略,以通过增强对暴露于不健康环境条件下的个体的抑制控制来集中力量预防向食物成瘾的过渡。

关键字:食物成瘾, 弱点, 恢复力, 操作式条件反射, 病毒载体法, 巧克力风味球, 强迫性, 冲动型

材料和试剂

用料
巧克力味颗粒(20毫克/粒,5TUL#1811223,TestDiet ,美国印第安纳州里士满)
微注射器(10μl ,Model 1701 N SYR,Cemented NDL,26 ga ,2 in,point style 3,#80039,Hamilton company,NV,USA)
聚乙烯管(PE-20,#C315CT,英国Plastics One)
双边引导插管(26号插管,在基座下方切割12毫米,#C235GS-5 / Spc,英国Plastics One)
双边内部套管(33英寸内部套管适合12 mm C235GS-5 / Spc ,突出3 mm,英国Plastics One)
渗透微泵(流量的0.25速率微升/小时,持续28天,2004模型,#0000298,的Alzet ,CA,USA)
手术刀(#02-036-040,AgnTho's ,Sweden)
手动钻(英国Plastics One DH-1)
钝头手术剪刀(#03-022-105,AgnTho's ,Sweden)
弯曲虹膜钳(#08-513-005,AgnTho's ,瑞典)
缝合线夹(#08-922-125,AgnTho's ,Sweden)
手术夹(#22-620-007,AgnTho's ,Sweden)
缝合线(黑色编织丝,TB10,3/8 TRIANG 15 mm 4/0 90 cm,#55327-50U,LorcaMarín ,西班牙)
 
试剂种类
蒸馏水
乙醇70%
碘(Betadine,500 ml,#716720,西班牙MEDA Pharma SAU)
生理盐水(0.9%,250毫升,#999790.8,厄尔尼诺实验室,西班牙)
葡萄糖血清(GlucosaVet 5g / 100ml,#1248 ESP,B.Braun Vet Care,西班牙)
锡林之夜(5 g,#2919-PS-CM,Visufarma ,西班牙)
Blastoestimulina (1%,30g,#719385,阿尔米勒,西班牙)
Vetflurane (异氟烷,250 ml,#2199-ESP,Virbac ,西班牙)
用0.9%无菌盐水(5 mg / ml)稀释的氯氮平N-氧化物(CNO,25 mg,#BML-NS105-0025,Enzo Life Sciences,NY)
麻醉剂
盐酸氯胺酮(75毫克/千克体重,10毫升,Ketamidor ,#580393 ,Richterpharma AG,奥地利)溶于0.9%无菌生理盐水
盐酸美托咪定(1 mg / kg体重,#570686,Domtor ;西班牙Esteve )溶于0.9%无菌生理盐水
溶解于0.9%无菌生理盐水中的盐酸阿替米唑(2.5 mg / kg体重,#570559,还原剂;西班牙Virbac )
庆大霉素(1 mg / kg体重,#999037,Genta-Gobens ;西班牙Laboratorios Normon )溶于无菌的0.9%生理盐水
美洛昔康(2 mg / kg体重,Metacam;#059/02 / 08CVFPT,勃林格殷格翰,莱茵)溶于0.9%无菌生理盐水
病毒载体(在-80 °C下储存)。例子
AAV8-HSYN-DIO-hM4D(GI) -的mCherry (1.21E + 13 GC / ml时,病毒载体生产装置Universität大学自治大学巴塞罗那)
AAV8-hSyn-DIO-mCherry(1.19E + 13 gc / ml,巴塞罗那自治大学病毒载体生产单位)
AAVrg pmSyn1-EBFP-Cre(6 x 10 12 v g / ml ,添加基因,病毒制备#51507-AAVrg)
 
设备
 
鼠标操作自我管理箱(型号ENV-307A-CT,Med Associates,乔治亚州,佛蒙特州,美国)
操作室配备有两个可伸缩杆(#ENV-312-2M,Med Associates),一个随机选择为活动杆,另一个作为非活动杆。按下主动杆会导致食物颗粒的输送与位于主动杆上方的刺激光(关联提示,#ENV-321M,Med Associates)配对,而按下被动杆则没有任何后果。两个杠杆之间的距离相等的食物分配器(#ENV-303M颗粒接收器,#ENV-203M-20,模块化颗粒分配器,Med Associates)允许在需要时传送食物颗粒。腔室的地板是网格地板(#ENV-307A-GF,Med Associates),用于在电击测试中传递电足电击,并在次日的电击诱导抑制中作为上下文提示。冲击测试。在其余的自我管理过程中,将带孔的金属板放置在网格地板上方。因此,小鼠可以区分不同的环境。室内照明灯放置在试验箱的天花板上(#ENV-315M,Med Associates)。这些腔室由铝和丙烯酸制成,并装在装有风扇的隔音和光衰减盒中,以提供通风和白噪声。(图1 )。
 
 
图1。一个自我管理室的示意图和图像。A.操作室示意图。操作室配备有两个可伸缩杆(活动和非活动),刺激灯和食物颗粒分配器。腔室的地板是网格地板,在电击测试期间会产生电脚电击,但在其余的自我管理阶段会被带孔的金属板覆盖。B.带有细节的面板图像,包含通过提示灯发出信号的主动杆和带有孔的金属板。C. Skinner Box的总体视图,其中包括操作箱灯,主动杆,提示灯,传递电击脚的网格地板,带孔的金属板和食品分配器。
 
立体定位仪(100微米分辨率,型号900,Koft仪器,美国C. A. )
站立式放大镜(OPMI 1 FR,卡尔·蔡司,美国)
微注射泵(PHD 2000,#MA1 70-20xx,哈佛仪器,霍利斯顿,MA,USA)
动物修剪器(#M630 ,西班牙Artero )
冷光(西班牙Leica Microsystems的Leica CLS 150x)
加热垫(#N2P 220-230,60W,50Hz,Daga ,西班牙)
热珠灭菌器(FST 250,#18000-45,AgnTho's ,Sweden)
 
软件
 
Med-PC软件(美国Med Associates Inc)。将所有行为记录在操作性自我管理室中的软件
GraphPad Prism软件(美国GraphPad软件)执行所有图形
SPSS软件(IBM,版本25)执行统计数据分析
 
程序
 
将雄性小鼠单独饲养在温度(21±1 °C)和湿度(55±10%)-随意控制食物和水的实验室条件。小鼠中的反向光周期的黑暗阶段测试(在8.00灯熄灭上午和8.00日下午)。
 
A.自我管理会议      
通过在前3 s内打开放置在腔室天花板上的室内灯,可以发出每次自我管理会话开始的信号。在长期食物成瘾方案中,由巧克力味小丸维持的每日自我给药持续时间为1小时,在短期食物成瘾方案中持续2小时,以增加每天可口颗粒的暴露量,以确保形成类似成瘾的表型。自我管理课程由2个药丸周期(25分钟和55分钟)和无药丸周期(10分钟)隔开。在沉淀期间,在主动响应与刺激光(提示光)配对后,沉淀被连续输送。每次输送颗粒后,将建立10 s的超时时间,此时提示灯熄灭,并且在对活动杆作出响应后未提供加强件。记录主动杆上的响应以及在超时期间执行的所有响应。在无药丸期间,没有药丸被递送,并且通过整个自给药室的照明来发出信号。每次训练后,将小鼠放回它们的家笼中。
  在操作性调节阶段中,小鼠按固定比例1(FR1)进行强化(1次按压力可产生1个小球),然后将FR增加至5(FR5)(5次按压力可产生1个小球) )的其余会话。如前所述(Martín-García等,2011),当满足以下所有条件时,即可获得实现操作员应答的标准:(1)小鼠保持稳定的应答,与应答的偏差小于20%。连续3个工作日赚取的辅助人员总数的平均值(稳定性的80%);(2)至少75%响应主动杆;(3)每节至少5个加固物。
B.衡量三类食物成瘾标准      
如所描述的(Mancino等,2015;Domingo-Rodriguez等,2020 ),使用三种行为测试来评估类似食物成瘾的标准,并从大鼠的可卡因成瘾样(Deroche-Gamonet等, 2004)。这三个标准总结了DSM-5中规定的基于DSM-IV的成瘾特征(Piazza and Deroche-Gamonet,2013),现在已通过YFAS 2.0纳入了食物成瘾诊断(Gearhardt等,2016)。
1.对反应的持久性(图2 A):在无丸剂阶段(10分钟)内无增强的主动响应(当盒子被照亮并发出无法传递颗粒的信号时),作为对食物寻求行为的持久性进行了测量。在进行性比率之前的连续3天中,对小鼠进行评分。      
2.动机(图2 B):增强的渐进比例表用于评估巧克力味颗粒的动机。赢得单个颗粒所需的响应根据以下系列逐步升级:1、5、12、21、33、51、75、90、120、155、180、225、260、300、350、410、465、540 ,630,730,850,1 ,000,1 ,200,1 ,500,1 ,800 2 ,100 2 ,400 2 ,700 3 ,000,3 ,400,3 ,800 4 ,200 ,4 ,600,5 ,000,和5 ,500的响应的最大数目,该动物进行以获得一个粒料是完成最后一个事件,被称为断裂点。进行比例实验的最大持续时间为5小时,或直到小鼠在1小时内对任何杠杆无反应为止。      
3.强迫性(图2 C):当每次递送的药丸与惩罚相关联时,在冲击试验期间(50分钟)的总冲击次数用于评估类似强迫性的行为,以前称为抵抗阻力(Deroche -Gamonet等,2004; Mancino等,2015)。小鼠被放置在一个自我管理的房间里,没有金属板有孔,因此暴露了网格地板(上下文提示)。在此电击过程中,小鼠在50分钟内处于FR5强化方案下,并且有2个方案变化:在第4个活动杠杆反应小鼠上,仅接受电击脚(0.18 mA,2 s)而没有沉淀。在第5个主动杠杆响应时,小鼠会收到另一个电击脚,电击脚带有巧克力味的颗粒和提示灯。如果小鼠在1分钟内未执行第5次响应,则在10 s的颗粒输送(超时时间)后和第4次响应后重新启动计划。      
 
图2。3种成瘾标准。一。坚持回应。无药丸期间(10分钟)内无增强的主动响应(当盒子被照亮并发出无法传递药丸的信号时)用于评估觅食的持久性。乙。动机。渐进比率测试用于评估巧克力味颗粒的动机。获得单个颗粒的响应呈指数增长。动物获得一个颗粒所执行的最大响应数是完成的最后一个事件,称为断点。Ç 。强迫性。当每次发射的药丸与惩罚相关联时,在冲击试验中的冲击总数用于评估类似强迫性的行为。
 
4. 3种成瘾性标准的归因(图3 ):在执行了3种行为测试以测量食物成瘾性行为后,小鼠在早期被归类为易感或适应力强的动物,而在成瘾性或非成瘾性动物中被归类。后期,取决于它们分别在早期或晚期达到的积极标准的数量。当特定行为测验的分数等于或高于巧克力对照组正态分布的第75个百分位数时,该动物被视为对成瘾状标准呈阳性。达到2或3个成瘾样标准的小鼠被视为易感动物,或被成瘾的动物和达到0或1个成瘾样标准的小鼠被视为有弹性或不成瘾的动物。      
注意:Ť他在本节中给出的图表示例与CBL57 / 6N小鼠获得的数据。数据来自Domingo-Rodriguez等人,2020年
 
图3。3种成瘾标准的归因。小鼠进行3项行为测试,以测量类似食物成瘾的行为,并针对每个标准获得单独的分数。在每个标准(水平虚线)中,巧克力对照组的正态分布的第75个百分位数被确定为一个阈值,当动物的个体得分等于或高于第75个百分位数时,认为该成瘾状标准的动物为阳性。达到2或3个成瘾状标准的小鼠被视为成瘾的动物,而达到0或1个成瘾状标准的小鼠被视为非成瘾的动物。在图中以四只小鼠(AD)为例。鼠标A给出的每个标准的值均低于达到0个标准的阈值,并被归类为非成瘾动物。鼠标B在持续性得分上显示出高于阈值的得分,低于动机和强迫性得分,达到1条标准,被归类为非成瘾动物。小鼠C在响应和强迫性方面的得分高于阈值,在动机上得分低于2,并且达到2个标准,被归类为上瘾的动物。鼠标d示出了得分的75以上每个标准的第百分实现3个标准,并且被分类为嗜动物。数据表示为单个值,中间值表示四分位间距。白色圆圈:0个标准的小鼠。绿圈:具有1个标准的小鼠。蓝色圆圈:具有2个条件的小鼠。红色圆圈:具有3个标准的小鼠。数据来自Domingo-Rodriguez等。,2020年。
 
C. 4种表型特征的测量被认为是容易上瘾的行为的因素      
1.冲动性(图4 A):在每次药丸递送后的超时时间段(10 s)内,非增强的主动反应被测量为类似冲动性的行为,表明一旦启动便无法停止响应(Logan等。,1997 ; Koob和沃尔科夫,2010) 。该标准考虑连续比率测试之前的连续3天。        
2.认知灵活性(图4 B):用一项逆转测试进行衡量,该测试表明了对先前已预测奖励可得性的刺激做出响应的能力(Schoenbaum等人,2011)。逆转试验是一个标准TR癌宁自我管理会话,但是活性和非活性杠杆被颠倒。        
3.竞争性联想学习(图4 C):通过提示诱发的寻食测试进行测量。提示导致的觅食时间持续90分钟,分为两个阶段:60分钟+ 30分钟。在最初的60分钟内,不会增强所有压杆的压力(活动和不活动的压杆均不会产生预定的后果)。在随后的30分钟中,根据FR5,先前在正常自我管理过程中与颗粒输送相关的白色提示灯会持续点亮30分钟。为了指示日程安排的变化,提示灯不连续地出现两次,持续4 s。        
4.厌恶联想学习(图4 D):用冲击诱发的抑制测试进行测量。在电击诱发的抑制测试中,将小鼠放在自我管理室中50分钟,并在电击测试中使用相同的网格地板。但是,在此FR5自我管理过程中,按下主动杆不会产生任何后果,不会产生冲击,不会产生巧克力味的颗粒,也不会发出提示。测量在这种震动诱发的抑制测试中非增强的主动反应,以进行厌恶联想学习。        
 
图4。将视为上瘾般行为的脆弱性因素4个表型性状。A.冲量是通过在每次药丸递送后的超时时间(10 s)内未与刺激光配对的非增强主动反应来测量的。B.认知能力通过逆转测试衡量。反向测试是FR5自管理会话,但是与之前的自管理会话相比,活动和非活动杠杆被颠倒了。Ç 。通过暗示诱导的食物寻找测试来衡量的竞争性联想学习。提示诱导的觅食测试是一项自我管理课程,需要90分钟,分为两个阶段:60分钟+ 30分钟。在最初的60分钟内,不会增强所有压杆的压力(活动和不活动的压杆均不会产生预定的后果)。在随后的30分钟中,根据FR5,与正常自我给药过程中的小丸递送相关的白色提示灯会持续点亮30分钟。d 。厌恶性联想学习通过冲击诱发的抑制测试测得。


D.实验设计      
小鼠食物成瘾的长期方案(图5 ):在长期食物方案中,在6个疗程中按照FR1强化方案训练小鼠,然后进行112疗程FR5,以自我给药巧克力味药丸。在FR5会议期间,3种上瘾样的标准(1)坚持反应(2),动机(3)和强迫性以及4种表型特征被视为容易上瘾的因素(1)冲动性(2)认知灵活性,(在每只小鼠的2个不同时间点评估3)竞争性联想学习和(4)厌恶联想学习。1日时间点是早期(会话FR5的1-18),和2次时间点是后期(会话FR5的95-112)。根据小鼠在早期达到的积极标准,将动物归类为有弹性的动物(0-1个标准)或脆弱的动物(2-3个标准),而在后期,将小鼠归为非上瘾性动物(0个标准) -1个标准)或成瘾的动物(2-3个标准)。
 
图5。长期食物成瘾小鼠模型实验序列的时间轴。在每天1小时的FR1强化训练下,对小鼠进行巧克力味药丸训练,持续6天,然后在FR5上训练112天。在FR5中,在早期和晚期考虑了2个时间点,以测量3种上瘾样的标准(对反应的持续性,动机和强迫性)。根据小鼠在早期达到的积极标准,将动物归类为有弹性的动物(0-1个标准)或脆弱的动物(2-3个标准),而在后期,将小鼠归为非上瘾性动物(0个标准) -1个标准)或成瘾的动物(2-3个标准)。在早期和晚期,还评估了4种表型性状作为成瘾易感性的因素(冲动性,认知灵活性,开性联想学习和厌恶联想学习)。
 
E.实验设计      
小鼠食物成瘾的简短规程(图6 ):在短期方案中,小鼠遵循长食物成瘾规程早期描述的相同行为程序,但由于手术病毒载体注射而有所不同。简短协议持续9周。在1日星期,训练小鼠以获取FR1(2次)和FR5(2次)加固的时间表下由巧克力味丸粒保持操作条件。在第二周,通过立体定向手术将目的病毒载体注射入小鼠。
病毒载体策略的示例:
化学发生方法(DREADD方法):是指在特定细胞类型中表达Cre重组酶的转基因小鼠确定的大脑区域中注射带有DREADD的Cre依赖性AAV 。
化学和逆行AAV联合疗法(逆DREADD方法):是指在确定的大脑区域中携带DREADD的Cre依赖性AAV和在其投射的大脑区域表达Cre重组酶以靶向Acre的AAV逆行组合注射。野生型小鼠体内的特定网络。
双边颅内注射后/ S的病毒载体/ s时,病毒载体的表达S是/期间4周(2的期间允许第二,3次,4次,和5个星期)。在此期间(3年初次周),小鼠FR5(4节)下恢复操作性响应的基础水平。在这4周(第5周)结束时,将填充有CNO或盐水的渗透微型泵皮下植入每只小鼠的背部。随后,将6期间日,7日,8日,和9个星期,当它是表示DREADD受体的长期CNO诱导的活化,小鼠是FR5下预定会议,随后的3瘾状标准测量。最后,根据小鼠所达到的积极标准,将动物分为非成瘾性(0-1个标准)或成瘾性动物(2-3个标准)。
 
图6 。短食成瘾小鼠模型实验序列的时间轴。在1日星期,训练小鼠以获取由巧克力味丸粒保持操作性条件 FR1(2次)和FR5下(3次)加固的时间表随后手术用于注射感兴趣的病毒载体(2 ND周)。手术后,病毒载体的表达被允许一段4周(2次,3次,4次,5次周)。在该期间(3开头RD周),小鼠是FR5(4次)下恢复的操作性响应的基础水平,并且在该期间(5的端第周),填充有CNO一个渗透性微型泵被植入。在6个,7个,8个和9个星期,当它是表示DREADD受体的CNO诱导的活化的慢性抑制,小鼠是FR5下预定会议,随后的3瘾状测定标准。
 
分步协议
 
长期食物成瘾小鼠方案
1.第1-6节(FR1):在这些会议中,采用了FR1加固时间表下的操作条件。        
2.课程1-112(FR5):在这些课程中,应用了根据FR5强化时间表进行的操作条件。        
会议1-18(FR5):FR5的这些会议构成了长期食物成瘾小鼠实验方案的早期阶段。
分会场3-5:应用FR5分会来评估反应持续性的标准和冲动的表型特征。
1)在无丸粒期(10分钟)内非增强的主动反应用于评估对反应标准的持久性。      
2)在每次药丸递送后的超时时间段(10 s)中未增强的主动反应用于评估冲动性表型性状。      
第6节:渐进比率测试,以评估激励标准。
第十节:冲击试验以评估强制性标准。
第11节:冲击诱发的抑制测试,以评估厌恶联想学习的表型特征。
第十五部分:提示诱导的觅食测试,以评估竞争性联想学习表型特征。
第18节:逆向测试以评估认知灵活性的表型特征。
根据早期达到的类似成瘾性阳性标准的数量,将小鼠分为易受攻击和有韧性的动物。
会话18-95(FR5):FR5会话。
第95-112节(FR5):这些会议构成了后期。
课程97-99:正常的FR5课程,以评估持续反应的标准和冲动的表型特征。
1)在无丸粒期(10分钟)内非增强的主动反应用于评估对反应标准的持久性。      
2)在每次药丸递送后的超时时间段(10 s)中未增强的主动反应用于评估冲动性表型性状。      
第100节:渐进比率测试,以评估激励标准。
104节:冲击试验以评估强制性标准。
第105节:冲击诱发的抑制测试,以评估厌恶联想学习的表型特征。
分会场109:提示诱发的食物寻找测试,以评估竞争性联想学习表型特征。
分会场112:进行逆向测试以评估认知灵活性的表型特征。
将成瘾和非成瘾动物中的小鼠分类,具体取决于后期达到的类似成瘾的阳性标准。在C57BL / 6N中使用此方案,我们获得了25%的成瘾小鼠。
 
短食成瘾小鼠方案
第一周:学习操作员的反应,以获取巧克力味的颗粒。
专场1-2:在FR1加固时间表下进行操作员调节。
专场3-4:在FR5加固时间表下进行操作员调节。
第2周:通过立体定向手术注射病毒载体。
立体定向手术方案(图7 )
注意:每次动物手术前,都要用热珠灭菌器对手术材料进行灭菌。
注射套管电路的准备。
卸下微注射器的柱塞(10 µl)。
将聚乙烯管(50厘米)连接到微注射器上。
使用注射器通过管子注入蒸馏水,填充管子和微注射器。检查水是否从微注射器的背面流出。
将一半的柱塞插入微注射器中。
将双侧内部套管(33号)的一侧连接到管路。按下微注射器柱塞并观察水如何从套管中流出而无泄漏,检查电路是否完好。
重复这些步骤与第二微量,其必须被附连到相同的内套管的第一的另一侧微量。
放置microsyringes在微注射泵。
吸出1 µl空气,在管子内形成气泡以监测微量注射。
吸出4 µl病毒载体(以前用冰除霜)。
立体定向器械的准备。
将双边引导套管放置并固定在立体定位支架的金属手柄中。
将连接到管道的双侧内部套管插入双侧引导套管中。双向引导插管用于保持内部插管笔直并很好地安装到固定器中,并在加载AAV之前找到前reg坐标。请注意,只有充满病毒载体的内部套管才可以穿透大脑。             
鼠标准备。
用盐酸氯胺酮和盐酸美托咪定混合并溶解在无菌的0.9%生理盐水中麻醉小鼠。腹膜内给药(分别为75 mg / kg和1 mg / kg体重)。
剃除鼠标头。
将鼠标放在立体定位设备中。使用耳棒和鼻夹检查头部是否固定。
在手术过程中,将加热垫放在鼠标下方,以保持适当的体温。
立体定向手术。
将碘涂在剃过的头上。
锡林之夜适用于眼睛,以避免角膜炎。
注意:必须使用立式放大镜完成以下步骤。
用手术刀在头中间进行垂直切割。
仔细刮擦头骨,以准确地显示前reg。
将套管放置在前reg上方,并注释前coordinates坐标。
计算中间横向,背腹和前后你的目标区域的坐标是注射到已被执行。
通过使用内侧-外侧,背侧-腹侧和前后位置的读数调整立体定位仪,将插管移动到特定的注射点。
用铅笔在颅骨上标记注射点。
按照铅笔标记,使用手动钻在头骨上打孔。
用生理盐水清洁注射区域。
将双侧套管插入孔中,并重新调整背腹坐标。
按照计算的坐标精确放置双侧插管。
在管路的两侧标记气泡,以监测进样情况。
进行病毒载体注射。
1)微量输液泵参数(例如前肢(PL)和伏隔核核心(NAc核心)区域的示例)      
目标体积:PL 0.2 µl ;NAc核心0.4 µl             
输液速度:PL 0.05 µl / min;NAc核心0.1 µl / min             
输液时间:4分钟
2)输注10分钟后,将套管留在原处,以防止回流。      
3)缓慢拔出套管10分钟。      
用生理盐水清洁注射区域。
用缝合线缝合切口。
在缝合的切口上应用blastoestimulina以促进真正的愈合。
皮下注射溶于无菌0.9%生理盐水的盐酸阿帕米唑(2.5 mg / kg体重)以逆转麻醉。
注射溶于无菌0.9%生理盐水的腹膜内庆大霉素(1 mg / kg体重)。
皮下注射美洛昔康(2 mg / kg体重)溶于无菌的0.9%生理盐水。
皮下注射葡萄糖血清(0.8 ml)。
将动物放在加热垫上,直到动物醒来。
在接下来的3个术后天中,每天检查小鼠并注射0.8 ml的葡萄糖血清。
清洁。
用乙醇清洁管道和套管,然后用蒸馏水和空气清洁。
用肥皂和水清洁所有手术材料。
双侧颅内注射后,需要4周(此方案中的第2、3、4和5周)才能正确表达病毒载体。
 
图7。立体定向手术。麻醉小鼠,并将其放入用于接受病毒载体颅内注射的立体定位设备中。所有注射均通过连接到聚乙烯的双侧注射套管进行。将注射器连接到注射针的聚乙烯管的长度内的置换气泡用于监控微量注射。通过使用微量输液泵以恒定的速率注射病毒载体。输注后,将注射套管再放置10分钟,以使液体扩散并防止回流,然后在10分钟内将其缓慢抽出。
注意:F经常检查聚乙烯管内部的气泡是否正确移位,以防止由于套管中的套管或烟气管阻塞而导致注入错误。
 
第三周:手术后进行手术调理以恢复基础的反应水平。
课程5-9:在FR5加固时间表下进行操作员调节。
第四周:操作调节。
第5周:在植入结束时,使用充满CNO或盐水的渗透微型泵。
渗透微型泵植入方案(图8 )
用异氟烷麻醉鼠标,并用胶带固定其前腿和后腿。
将鼠标刮在下背部。
用乙醇清洁刮过的下背部。
横切(1.5厘米),以后腿的起点为参考。
将皮肤与背部肌肉分开,通过切口引入钝头剪刀。
在背部中部引入先前充满CNO(用0.9%无菌生理盐水稀释:5 mg / ml )或盐水的渗透微型泵(ALZET 2004)。泵盖朝向动物的头部。
用手术夹闭合切口(约4个)。
在该区域涂抹碘酒。
 
图8。渗透微型泵植入过程的示意图。A.用异氟烷麻醉鼠标,并刮除下背部。用乙醇清洁刮毛区域。乙。横切(1.5厘米),以后腿的起点为参考。Ç 。将皮肤与背部肌肉分开,通过切口引入钝头剪刀。d 。在背部中部皮下引入先前充满CNO或盐水的渗透微型泵。泵盖朝向动物的头部。用外科手术夹闭合切口,并在该部位涂碘。Ë 。侧视图,显示动物在皮下植入的微型泵中清醒。                                                        
注意:手术后第二天检查微型泵的正确位置。如有必要,请在微型泵的放置部位进行平稳按摩,以重新定位并防止皮肤受伤。
 
第6周:在由CNO激活的病毒载体表达期间的操作条件。
课程10-14:在FR5加固时间表下进行操作员调节。
第7周:在由CNO长期激活的病毒载体表达过程中进行操作性调节。
分会15-20:在FR5加固时间表下进行操作员调节。
第8周:在由CNO长期激活的病毒载体表达过程中的操作条件。
第21节:FR5加固时间表下的操作员调节。
课程22-24:正常的FR5课程,评估对反应和冲动的持久性。
在无丸粒期(10分钟)内未增强的主动反应用于评估对反应标准的持久性。
每次药丸递送后的超时时间段(10 s)内未增强的主动响应被测量为类似冲动的行为。
第二十五节:渐进比率测试以评估动机标准。
第9周:在由CNO长期激活的病毒载体表达过程中的操作条件。
专场26-28:FR5加固时间表下的操作员调节。
第二十九课:冲击试验以评估强制性标准。
第30节:冲击诱发的抑制测试,以评估厌恶联想学习。
将成瘾和非成瘾动物中的小鼠分类,具体取决于获得的成瘾阳性标准的数量。在通过化学方法抑制PL-NAc核心途径的C57BL / 6J小鼠中使用该方案,我们获得了50%的上瘾小鼠。
验证病毒表达。
使用蠕动泵以每分钟30毫升的速度向蠕动泵以每分钟30毫升的速度向心内灌注4%多聚甲醛(PFA)的0.1 M Na 2 HPO 4 /0.1 M NaH 2 PO4缓冲液(PB)(pH 7.5)进行灌注。事先通过腹膜内注射氯胺酮/美托咪定的混合物(0.2毫升/ 10克体重)彻底麻醉动物。
提取大脑并用4%PFA固定24小时后,在4°C下将它们转移至30%蔗糖溶液中。
使用冷冻切片机制作冠状切片(  30μm ),并将其储存在4°C的5%蔗糖溶液中,直至进行免疫荧光研究。
使用针对所使用的病毒载体荧光报告基因的特异性抗体进行免疫荧光研究。
用共聚焦显微镜观察大脑的染色部分,以评价注射正确放置的组织学验证。
制作有代表性的免疫荧光图片(图9)。
 
图9 。代表性的免疫荧光照片显示在PL注射位点(左)和在NAc核心处的Cre重组酶(右)检测到Cre依赖的hM4Di-mCherry 。数据来源于Domingo-Rodriguez等。,2020年。
 
数据分析
 
排除标准
其余所有实验序列中排除了对所有FR5期的反应< 25%且未达到采集标准的动物。
在实验的脑区域之外具有病毒载体表达的动物被排除在实验之外。
 
图形和统计分析
加强件数量
图:每组在整个食物成瘾规程中,每个疗程中获得的强化剂的平均数量(图10)。平均值±SEM代表数据。
统计分析:方差分析分别对协议的每个步骤(FR1,FR5)进行重复测量,分析补强剂的数量,以测试随时间的演变。
 
图10 。对PL- NAc核心投射的化学生成抑制作用导致其对高度可口食品的强迫行为。在操作培训期间由巧克力味药丸维持的强化剂数量(平均值±SEM)。在白色盐水处理的小鼠和红色为CNO处理的小鼠(n = 12对于盐水处理的小鼠和n = 22,用于CNO治疗的小鼠)。
 
3种上瘾样标准的行为测试(对反应,动力和强迫性的持续性)
图表:这3个标准的数据表示为具有中值和四分位数范围的单个值(图11)。
对反应的持久性:无颗粒的连续3天每天10分钟内非增强主动反应总数的平均值。
动机:在渐进式比例测试的5小时内达到断裂点。
强迫性:在电击试验中,小鼠在50分钟内受到的电击次数,每次击球都与足部电击有关。
晚期的3种成瘾状标准的图表显示了一条水平虚线,表示对照组分布的第75个百分位数,被认为是对1个标准阳性的小鼠的阈值。
 
图11 。三种成瘾状标准的行为测试显示,经CNO处理的小鼠的强迫性增加,且具有PL- NAc核心通路的抑制作用(中位数和四分位数范围的个体值,U Mann - Whitney,** P < 0.01)。水平虚线表示用盐水处理的小鼠的分布的第75个百分点。用灰色填充圆圈表示上瘾的小鼠(用盐水处理的小鼠)和用红色表示的CNO处理的小鼠(用盐水处理的小鼠的n = 12,用CNO处理的小鼠的n = 22)。
 
统计分析
通过Kolmogorov-Smirnov正态性检验分析样品的分布。
如果样本未遵循正态分布(Kolmogorov-Smirnov正态性检验中的显着性值),则由U Mann - Whitney分析两组之间的比较。
如果样本遵循正态分布(在Kolmogorov-Smirnov正态性检验中无显着价值),则通过Student 's t检验分析两组之间的比较。
对成瘾和非成瘾动物中的小鼠进行分类
图:HO的零件LES图表示各组中嗜和非嗜动物的百分比(图12) 。
统计分析:通过卡方检验分析成瘾小鼠与未成瘾小鼠的百分比,将观察到的频率与对照组中获得的频率进行比较。
 
图12 。被归类为食物成瘾动物的PL- NAc核心途径小鼠抑制后,CNO处理的百分比增加(卡方,*** P < 0.001,盐水处理小鼠为n = 12 ,CNO处理小鼠为n = 22老鼠)。
 
每种成瘾状标准的个体价值与达到的阳性标准最终数量之间的相关性。通过皮尔逊相关系数分析相关性。
4个表型性状,以成瘾样行为(冲动,认知灵活性,食欲联想学习漏洞的因素的行为测试,和厌恶联想学习):
图形:4个表型性状的数据表示为具有中值和四分位数范围的单个值。
冲动性:每次送丸后连续3次每日超时(10 s)内50分钟内未增强主动反应的平均数。
认知灵活性:反转测试中60分钟内有效和无效反应的数量,与之前的基础训练相比,有效和无效杠杆被反转。
竞争性联想学习:
1)在提示诱导的食物寻找测试的60分钟内做出积极反应,在此期间未增强操纵杆压力,随后在30分钟内做出主动响应,在此期间主动操纵杆压力(FR5)与提示灯相关没有颗粒输送(平均值±SEM)。      
2)提示灯提示后,积极的反应有所增加。      
厌恶性联想学习:电击诱发抑制测试中50分钟内未增强的主动反应的数量。
统计分析
通过Kolmogorov-Smirnov正态性检验分析样品的分布。
如果样本未遵循正态分布(Kolmogorov-Smirnov正态性检验中的显着性值),则由U Mann - Whitney分析两组之间的比较。
如果样本遵循正态分布(在Kolmogorov-Smirnov正态性检验中无显着价值),则通过Student 's t检验分析两组之间的比较。
甲P -值< 0.05被用于确定统计显着性。
 
预期结果
注意:本节中显示的图形示例是将化学上抑制了PL-NAc核心途径的小鼠与未抑制该网络的小鼠进行比较所获得的数据。所有这些数据都发表在Domingo-Rodriguez等人的手稿2020中。
 
笔记
 
在整个短期和长期食物成瘾小鼠实验方案中,每周测量一次体重和食物摄入量。这些测量在使用成瘾的CNO渗透微型泵的短食物成瘾小鼠实验方案中尤其重要。我们证明,在我们的条件下,没有发现CNO对体重,食物摄入和运动能力有副作用(Domingo-Rodriguez等人,2020年)。
 
致谢
 
我们感谢E. Senabre和S. Kummer的批评家和技术支持。这项工作是由西班牙支持部:德Economía Ý Competitividad -MINECO(#SAF2017-84060-R-AEI / FEDER-UE),西班牙研究所德Salud的卡洛斯III,RETICS-RTA(#RD12 / 0028/0023),则Generalitat加泰罗尼亚,AGAUR(#2017年SGR-669),ICREA-学术界(#2015)和西班牙部:德Sanidad ,SERVICIOS Sociales ē IGUALDAD ,计划全国自我Drogas是(#PNSD-2017I068)到RM,Fundació拉Marató-TV3 (#2016 / 20-30)和将西班牙卫生部的国家Sobre Drogas计划(#PNSD-2019I006)到EM-G 。所描述的方法先前已在Domingo-Rodriguez等人中使用。,2020年。带有图纸的图形是通过BioRender.com创建的。
 
利益争夺
 
作者没有利益冲突。
 
伦理
 
由当地伦理委员会根据欧洲共同体理事会2010/63 / EU的指导方针进行所有实验方案及批准(科米特客位D'Experimentació动物的Parc de Recerca生物医药巴塞罗那,CEEA-PRBB,协议ñ9687 )。
参考文献
 
Deroche- Gamonet ,V.,Belin,D。和Piazza,PV(2004)。大鼠中成瘾样行为的证据。科学305(5686):1014-1017。
Domingo-Rodriguez,L.,Ruiz de Azua,I.,Dominguez,E.,Senabre,E.,Serra,I.,Kummer,S.,Navandar,M.,Baddenhausen,S.,Hofmann,C.,Andero ,R.,Gerber,S.,Navarrete,M.,Dierssen,M.,Lutz,B.,Martín-García,E。和Maldonado,R。(2020年)。特定的伏前核伏隔途径控制着韧性和对食物成瘾的脆弱性。Nat Commun 11(1):782。
Gearhardt,AN,Corbin,WR和Brownell,KD(2016)。耶鲁食品成瘾量表2.0版的开发。心理上瘾者行为30(1):113-121。
Hamer,D.(2002年)。遗传学。重新思考行为遗传学。科学298(5591):71-72。
Koob ,GF和Volkow,ND(2010)。成瘾的神经回路。神经心理药理学35(1):217-238。
Koob ,GF和Volkow,ND(2016)。成瘾的神经生物学:神经回路分析。柳叶刀精神病学3(8):760-773。
Lindgren,E.,Gray,K.,Miller,G.,Tyler,R.,Wiers ,CE,Volkow,ND和Wang,GJ(2018)。食物成瘾:与药物滥用有关的常见神经生物学机制。前BIOSCI (地标编辑)23:811-836。
Logan GD,Schachar RJ和Tannock R.(1997)。冲动与抑制控制。心理科学8(1):60-64。
曼奇诺,S.,Burokas ,A.,Gutierrez的-Cuesta的,J.,Gutierrez-马托斯,M.,马丁·加西亚,E.,Pucci的,M.,法尔科尼,A.,德达达里奥,C.,Maccarrone , M.和Maldonado,R.(2015)。通过饮食上瘾样行为促进表观遗传和蛋白质组表达的变化。Neuropsychopharmacology 40(12):2788-2800。
马丁加西亚,E.,Burokas ,A.,Kostrzewa ,E.,Gieryk ,A.,Korostynski ,M.,Ziolkowska ,B.,Przewlocka ,B.,Przewlocki ,R。和Maldonado的,R。(2011)。恢复小鼠觅食行为的新操作模型。心理药物学(Berl )215(1):49-70。
穆尔,CF,萨比诺,五,Koob ,GF和Cottone ,P.(2017)。病理性暴饮暴食:强制性构造的新证据。神经心理药理学42(7):1375-1389。
Nestler ,EJ,Pena,CJ,Kundakovic ,M.,Mitchell,A.和Akbarian ,S.(2016)。心理疾病的表观遗传基础。神经科学家22(5):447-463。
Piazza,PV和Deroche- Gamonet ,V.(2013)。向成瘾过渡的多步骤一般理论。心理药物学(Berl )229(3):387-413。
Pursey ,KM,Stanwell,P.,Gearhardt ,AN,Collins,CE和Burrows,TL(2014)。由耶鲁大学食物成瘾量表评估的食物成瘾患病率:系统评价。营养素6(10):4552-4590。
Schoenbaum,G.,Roesch,MR,Stalnaker,TA和Takahashi,YK(2011)。眶额皮质和预期结果:优化行为和感觉知觉。于:Gottfried ,J 。一。(E d 。) 。感觉和奖励的神经生物学。博卡拉顿(FL):CRC出版社/泰勒和弗朗西斯。
登录/注册账号可免费阅读全文
  • English
  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2020 The Authors; exclusive licensee Bio-protocol LLC.
引用:Martín-García, E., Domingo-Rodriguez, L. and Maldonado, R. (2020). An Operant Conditioning Model Combined with a Chemogenetic Approach to Study the Neurobiology of Food Addiction in Mice. Bio-protocol 10(19): e3777. DOI: 10.21769/BioProtoc.3777.
提问与回复
提交问题/评论即表示您同意遵守我们的服务条款。如果您发现恶意或不符合我们的条款的言论,请联系我们:eb@bio-protocol.org。

如果您对本实验方案有任何疑问/意见, 强烈建议您发布在此处。我们将邀请本文作者以及部分用户回答您的问题/意见。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片的形式来说明遇到的问题。

如果您对本实验方案有任何疑问/意见, 强烈建议您发布在此处。我们将邀请本文作者以及部分用户回答您的问题/意见。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片的形式来说明遇到的问题。