TYPE Methods PUBLISHED 15 December 2022 DOI 10.3389/fnbeh.2022.1046259 Protocol to assess rewarding OPEN ACCESS brain stimulation as a learning EDITED BY Ana Cicvaric, Albert Einstein College of Medicine, and memory modulating United States REVIEWED BY treatment: Comparison Dadasaheb Mahadeo Kokare, Rashtrasant Tukadoji Maharaj Nagpur University, India between self-administration and Andrew Charles Harris, University of Minnesota Twin Cities, United States experimenter-administration *CORRESPONDENCE Soleil García-Brito Laia Vila-Solés 1 , Soleil García-Brito 1*, soleilcristina.garcia@uab.cat Laura Aldavert-Vera 1 , Elisabet Kádár 2 , SPECIALTY SECTION This article was submitted to Gemma Huguet 2 , Ignacio Morgado-Bernal 1 and Emotion Regulation and Processing, Pilar Segura-Torres 1 a section of the journal 1 Frontiers in Behavioral Neuroscience Departament de Psicobiologia i de Metodologia de les Ciències de la Salut, Institut de Neurociències, Universitat Autònoma de Barcelona, Barcelona, Spain, 2 Departament de Biologia, RECEIVED 16 September 2022 Universitat de Girona, Girona, Spain ACCEPTED 25 November 2022 PUBLISHED 15 December 2022 CITATION Vila-Solés L, García-Brito S, Aldavert-Vera L, Kádár E, Huguet G, Intracranial electrical self-stimulation (ICSS) is a useful procedure in animal Morgado-Bernal I and Segura-Torres P research. This form of administration ensures that areas of the brain reward (2022) Protocol to assess rewarding brain stimulation as a learning system (BRS) are being functionally activated, since the animals must perform and memory modulating treatment: an operant response to self-administer an electrical stimulus. Rewarding Comparison between self-administration post-training ICSS of the medial forebrain bundle (MFB), an important system and experimenter-administration. of the BRS, has been shown to consistently improve rats’ acquisition and Front. Behav. Neurosci. 16:1046259. doi: 10.3389/fnbeh.2022.1046259 retention in several learning tasks. In the clinical setting, deep brain stimulation (DBS) of different targets is currently being used to palliate the memory COPYRIGHT © 2022 Vila-Solés, García-Brito, impairment that occurs in some neurodegenerative diseases. However, the Aldavert-Vera, Kádár, Huguet, stimulation of the MFB has only been used to treat emotional alterations, Morgado-Bernal and Segura-Torres. This is an open-access article not memory disorders. Since DBS stimulation treatments in humans are distributed under the terms of the exclusively administered by external sources, studies comparing the efficacy Creative Commons Attribution License (CC BY). The use, distribution or of that form of application to a self-administered stimulation are key to reproduction in other forums is the translationality of ICSS. This protocol compares self-administered (ICSS) permitted, provided the original author(s) and the copyright owner(s) and experimenter-administered (EAS) stimulation of the MFB on the spatial are credited and that the original Morris Water Maze task (MWM). c-Fos immunohistochemistry procedure was publication in this journal is cited, in carried out to evaluate neural activation after retention. Results show that accordance with accepted academic practice. No use, distribution or the stimulation of the MFB improves the MWM task regardless of the form reproduction is permitted which does of administration, although some differences in c-Fos expression were found. not comply with these terms. Present results suggest that MFB-ICSS is a valid animal model to study the Frontiers in Behavioral Neuroscience 01 frontiersin.org Vila-Solés et al. 10.3389/fnbeh.2022.1046259 effects of MFB electrical stimulation on memory, which could guide clinical applications of DBS. The present protocol is a useful guide for establishing ICSS behavior in rats, which could be used as a learning and memory- modulating treatment. KEYWORDS intracranial self-stimulation, medial forebrain bundle (MFB), brain reward system, learning, memory GRAPHICAL ABSTRACT Comprehensive timeline of an experiment using self-administered or experimenter-administered intracranial stimulation to modulate learning and memory. Introduction self-stimulation (ICSS), formerly described by Olds and Milner (1954). Motivational and reinforcement processes are directly Medial forebrain bundle-intracranial electrical self- involved in operant conditioning (Bamford et al., 2018). stimulation, especially when administered post-training, is However, data from many animal studies have demonstrated capable of improving the consolidation of both non-declarative that the stimulation of regions belonging to the brain (Aldavert-Vera et al., 2013; García-Brito et al., 2017) and reward systems (BRS) also modulates learning beyond operant declarative memory (Chamorro-López et al., 2015; García-Brito conditioning, by acting as a facilitating treatment for different et al., 2020), and even promoting the functional recovery of learning and memory tasks (Morgado-Bernal and Segura- induced memory deficits (Segura-Torres et al., 2010; Kádár Torres, 2022). One of the main regions of the BRS, and one of the et al., 2014). ICSS of the MFB also manages to activate plasticity most commonly targeted is the medial forebrain bundle (MFB) processes in different circuits involved in several types of in the lateral hypothalamus (Milner, 1991). Animals implanted memory (Aldavert-Vera et al., 2013; Kádár et al., 2014; Huguet with electrodes to the MFB self-administer the stimulation et al., 2020; Puig-Parnau et al., 2020). Despite the promising trains, due to the reinforcing nature of the stimulation. This results in animal models, clinical trials using deep brain kind of administration paradigm is known as intracranial stimulation (DBS) to alleviate memory deficits in disorders such Frontiers in Behavioral Neuroscience 02 frontiersin.org Vila-Solés et al. 10.3389/fnbeh.2022.1046259 as Alzheimer’s disease usually target discrete memory-related in order to assess motivational states, using ICSS behavior as a regions (i.e., fornix, entorhinal cortex, or Basal nucleus of dependent variable. Meynert) (Lyons, 2011; Aldehri et al., 2018), rather than the Given that basic research using animals can sometimes bring MFB or any other BRS regions. On the other hand, the MFB about unexpected or even potentially harmful consequences, is currently being used as a therapeutic target in disorders it is of paramount importance to establish well-characterized such as anhedonia and depression (Bewernick et al., 2017; protocols in order to obtain reliable results with minimum Fenoy et al., 2018). Taken all together, these results suggest that impact to the animals’ welfare. On that basis, a comprehensive targeting MFB fibers could be useful in the treatment of diseases list of the most frequent issues and troubleshooting is provided. presenting diverse symptomatology, including emotional, All procedures were carried out in compliance with the motivational and memory alterations, which could be present Directive 2010/63/EU and were approved by the institutional in Alzheimer’s disease (Bennett and Thomas, 2014; Wei et al., animal care committee. 2020). While ICSS is a procedure in which animals self-administer the electrical stimulation, DBS in humans is not self- Materials and equipment administered. Thus, in order for animal research to inform future clinical studies, it is important to determine whether Stimulation electrode and ground the desired therapeutic effects can be achieved by either form screw materials of administration. The primary goal of this protocol is to compare self- Materials shown in Figure 1. administered (ICSS) and experimenter-administered (EAS) stimulation of the MFB in terms of its effectiveness in a. General instruments (pliers, file, ruler, scissors) enhancing spatial memory consolidation. In order to do this, b. Scalpel the animals are trained in a hippocampus-dependent spatial c. Saw task in the Morris Water Maze (MWM) (Vorhees and Williams, d. Soldering iron 2014b), and receive a post-training treatment (ICSS or EAS) e. Swivel bench vise immediately after each of the 5 MWM acquisition sessions. f. Insulated stainless steel electrode wire The effects of the stimulation of the MFB are evaluated g. Female connector every 24 h in the acquisition sessions, and in the retention h. Screws test 72 h after the last acquisition session. In addition, the i. Brush and solder flux paste neural activation in different brain regions was evaluated j. Tin wire 90 min after retention, using a c-Fos immunohistochemistry k. Copper wire protocol. l. Power supply The secondary goal of this protocol is to serve as a m. Petri dish with water comprehensive how-to guide in the establishment of consistent n. Sodium chloride ICSS behavior in rats, in order to use it as a treatment to study o. Spoon its relationship to learning and memory. We provide detailed p. Male pin explanations for the preparation of homemade electrodes, electrode implantation surgery, and procedures to obtain self- administration behavior. Moreover, we specify the parameters Surgery equipment for the use of two forms of rewarding stimulation administration (ICSS and EAS) as a treatment to improve performance in Surgery equipment shown in Figure 2. a spatial task in the MWM. Although this protocol describes the post-training administration of the treatment, some aspects a. Paper towel could also be applied to the design of experiments aimed at b. Hair trimmer assessing the effects of MFB stimulation in other phases of c. Artificial eye drops learning and memory. The procedure and outcome for the d. Brush learning and memory task in the MWM as well as for the c-Fos e. Heating pad expression immunochemistry are also included in the protocol. f. Surgical lights with flexible arms Moreover, the specific steps to establish ICSS behavior safely g. Digital display of coordinates and effectively may also be of interest to researchers working in h. Gas extractor fields of study in which reward is involved, such as addictive i. Surgical instrument (scalpel, spatula, x4 curved mosquito behaviors (Baird et al., 2021). Carlezon and Chartoff (2007) forceps, scissors, x1 straight mosquito forceps, standard provide a more detailed description of the procedure to follow forceps) Frontiers in Behavioral Neuroscience 03 frontiersin.org Vila-Solés et al. 10.3389/fnbeh.2022.1046259 FIGURE 1 Setup of the working area with instruments needed to build electrodes and ground screws. (A) Materials and tools used for the preparation of stimulation electrodes and ground screws. (B) Magnification of some of the materials needed for this process. (C) Power supply and materials used to check electrode current. (D) Male pin used to attach the electrode prepared to the power supply. j. Lidocaine Electrical brain stimulation equipment k. Iodine l. Meloxicam Skinner box m. Hydrogen peroxide For all experimental conditions (ICSS, EAS, or non- n. Cotton swabs stimulated animals), a conventional 25 × 20 × 20 cm Skinner o. Pencil box with a lever inserted in one of the walls was used, as shown p. Sterile 0.9% saline solution in Figure 3. q. Surgical drill r. Electrodes (previously prepared) s. Screws and ground screws (previously prepared) Stimulator t. Acrylic cement (mixture) The stimulator used for these experiments is the model u. Suture materials CS2-10 (previously the model CS20 IM) from Cibertec, v. 21G needle, indented tip which creates trains of bipolar sinusoidal waves at a constant w. Sterile-absorbable hemostatic gelatin (Cutanplast ) R current at 50 Hz (see Figure 4B). The output stimulation Frontiers in Behavioral Neuroscience 04 frontiersin.org Vila-Solés et al. 10.3389/fnbeh.2022.1046259 FIGURE 2 Picture of equipment and surgery area. (A) Stereotaxic instrument with labeled materials. (B) Instruments and materials. (C) Magnification of the male pin attached to the stereotaxic arm. The female connector of the electrodes fabricated previously is attached to this male pin for support. FIGURE 3 Picture of Skinner box used and details. (A) Clear door Skinner box used for intracranial electrical self-stimulation (ICSS)/experimenter-administered stimulation (EAS) administration. Note that there is not a high level of lighting. This is due to standardized behavioral testing conditions from our lab. (B) Male pin is used to connect the stimulator to the female entrance of the stimulation electrode. Note that acrylic cement is used to fix the male pin to the cable connected to the stimulator. (C) The wire connected to animals’ female pin is attached to the electrode swivel located in the Skinner box ceiling order to provide rotational movement to animals. circuit is isolated optoelectronically. The duration of the that prevents unwanted stimulations. Before reaching the trains can be adjusted (in this case we use 0.3 ms). The stimulator, the triggered signal goes through a circuit of triggering of the train can be performed manually by a pulse inhibition, which can be adjusted to different times. button or a response lever inside the Skinner box. The The goal of this system is to not let another signal go triggering signal is applied into an anti-rebound circuit through until the preset inhibition time has passed. The Frontiers in Behavioral Neuroscience 05 frontiersin.org Vila-Solés et al. 10.3389/fnbeh.2022.1046259 FIGURE 4 Stimulator used for this experiment. (A) Picture of the stimulator with its main features depicted. (B) Illustration depicting the stimulator circuit. stimulator has two digital displays, the Responses, in which South-west (SW): teddy bear and a white plastic cylinder with all responses are marked, and the Reinforcements, in which horizontal black stripes, and North-west (NW): cross cutout in all stimuli received by the animal are shown, as shown in a box with a light inside, as observed in Figures 5B, C. Figure 4A. Swim paths were recorded using a closed-circuit video camera (Smart Video Tracking System, Version 2.5, Panlab) with a wide-angle lens 1.75 m above the center of the pool. See Table 1 for all reagents and resources with their source Behavioral apparatus and identifiers. The behavioral apparatus used in this experiment to test spatial learning and memory was the Morris Water Maze (MWM). The MWM is a maze in which subjects have to find Methods a submerged platform in the middle of the four quadrants of the pool. The tank is virtually divided into four quadrants, Stimulation electrode and ground defining four cardinal points: North (N), South (S), East (E), screw preparation and West (W), as observed in Figure 5A. Subjects have to identify and use the spatial cues hung around the pool in order Timing: 2 h (2 h/6 electrodes and 6 ground screws). to find the platform and escape the pool. The cues used in this Important considerations: Always prepare the electrodes experiment are; North-east (NE): beach ball with different color between 1 and 3 days prior surgery. This time period stripes, South-east (SE): indirect light of a lamp in the curtain, should be taken into account to prevent oxidation of the Frontiers in Behavioral Neuroscience 06 frontiersin.org Vila-Solés et al. 10.3389/fnbeh.2022.1046259 FIGURE 5 Morris Water Maze apparatus. (A) Illustration depicting the spatial Morris Water Maze configuration used in our lab. (B) Picture of the objects used in the South-east (SE) and South-west (SW) quadrants. (C) Picture of the objects used in the North-west (NW) and North-east (NE) quadrants. stimulation electrode. Gloves may complicate most steps of this a magnifying glass if all the insulation has been correctly procedure; instead, wash your hands carefully before starting removed. and after finishing. 6. Place the stripped end of the electrode wire on the base made by the rolled wire. While holding the electrode, take 1. Prepare all material necessary for this procedure in one of the extremes of the wire and roll it around the advance. Turn on the soldering iron. electrode wire and the horn of the base 3–4 times in order 2. Prepare 2-bases section using a saw. After sawing the to secure the electrode to the horn (see Figure 6E). Once sections, file any imperfections. Caution: Carefully check it is attached, cut the excess copper wire at both ends and if the sawing has exposed any part of the metallic horns solder them. Important: the insulated end of the electrode inside the base. If so, discard and obtain a new section. wire should end up facing us, as shown in Figures 6H–K. 3. Place the base in the swivel bench vise and secure it. Cut 7. Once we have finished preparing our stimulation electrode, 7 cm of copper wire. Leave about 4 cm of wire to pull at it must be functionally checked. Connect the base to a the base and roll up the rest of the wire around the horn power supply attachment piece, as shown in Figure 6F. until it is completely covered. Leave around 1 cm excess First, attach the power supply to the part of the base wire at either end of the horn and cut the wire excess at where the electrode wire is located. Introduce the tip of the the opposite end. Use the brush to collect some solder flux electrode wire into the solution of salted water (avoid the paste and cover the horn with it. Take the soldering iron, copper wire touching the water), as shown in Figure 6G. take a drop of tin and cover the horn (see Figures 6A–C). If electrical current is running through the stimulation Caution: both the flux paste and the tin should always be electrode, you should be able to see bubbles around the tip applied from the top to bottom. If done in the opposite of the electrode wire and the power supply should indicate direction, the copper wire could be dislodged. an increase in current levels. Repeat the same process for 4. Cut another 7 cm length of copper wire. Place the center of the copper wire (ground). it behind the other horn and roll it around the horn once 8. Check if the remaining length of the electrode to create a stable base. wire is 1 cm long. 5. Cut a 1.3 cm insulated electrode wire (Figure 6D). Once 9. Store the stimulation electrodes in a sealed box until use. it is cut, scratch off 3–4 mm of one of the extremes using 10. To prepare the ground screw, place the screw in the swivel a scalpel in order to strip the insulating layer. Check with bench vise with its head facing downward and secure it. Frontiers in Behavioral Neuroscience 07 frontiersin.org Vila-Solés et al. 10.3389/fnbeh.2022.1046259 TABLE 1 Description of all reagents (subjects, chemicals, solution TABLE 1 (Contiunued) preparations, other relevant tools, and software) used in this protocol with their corresponding source and identifiers. Reagent or Source Identifier resource Reagent or Source Identifier resource Other Scalpel blade Centauro Ref. 1110910V Subjects www.centauro.es Wistar rats Charles River In this experiment, the 200 µm Bilaney Ref. 008SW/30S www.criver.com offspring of the animals diameter www.bilaney.com bought were used to insulated avoid stress effects due to stainless steel transportation. electrode wire– Chemicals straightened for Solder flux paste Shoptronica Ref. 0689593987280 30 cm www.shoptronica.com Female Diotronic S.A. Ref. SSQ36SSTG Sodium chloride Merck, Sigma Aldrich Ref. 1064040500 connector for www.diotronic.com www.sigmaaldrich.com electrode base Artificial eye drops Centauro Lubrithal 10gr Male pins Diotronic S.A. Ref. SSQ36SSTG www.centauro.es Ref. 5701170313291 2.54 mm www.diotronic.com Lidocaine Royal Dent Ref. 12408 Screws Harvard Apparatus Ref. CMA7431021 https://www.royal-dent.com/ www.harvardapparatus.com Tin wire Diotronic S.A. Ref. HIF00651 Iodine Centauro Ref. 8413204701031 www.diotronic.com www.centauro.es Copper wire Diotronic S.A. Ref. WIK01N Metacam Centauro Ref. 4028691555438 www.diotronic.com www.centauro.es Power supply Discontinued Model SK112 Hydrogen peroxide Centauro Ref. 8410088000144 (20V/0.2A) www.centauro.es Stereotaxic Cibertec https: Intravenous serum Centauro Ref. 4030539076609 equipment www.cibertec.es //www.cibertec.es/es/ www.centauro.es catalogo-de-productos/ Acrylic cement Proclinic Solid resin (1 kg): bydiscipline/88 www.proclinic.es Ref. AVCPP0801000 Sterile- Uppermat Ref. CU101024 Liquid resin (1000 ml): absorbable www.uppermat.com Ref. AVCPV01000 hemostatic Isoflurane Centauro Ref. 8436025713298 gelatin www.centauro.es (Cutanplast) TRIS Serva Ref. 37190.02 Anesthesia Cibertec https: www.serva.de/enDE equipment www.cibertec.es //www.cibertec.es/es/ Sodium chloride PanReac AppliChem Ref. 131659.1210 catalogo-de-productos/ www.itwreagents.com/ bydiscipline/37 Bovine Serum EMD Millipore Corp. Ref. 12650-250GM Skinner box Cibertec https: Albumin (BSA) www.merckmillipore.com/ www.cibertec.es //www.cibertec.es/es/ catalogo-de-productos/ Triton X-100 Merck Group Ref. X100-500 ml bydiscipline/91 www.sigmaaldrich.com Stimulator Cibertec Made to experiment Mouse anticFos Santa Cruz Biotechnology Ref. 166940 model CS2-10 www.cibertec.es specification. primary antibody www.scbt.com Software Biotinylated goat Jackson Immunoresearch Ref. 115-065-166 anti-mouse-IgG www.jacksonimmuno.com Smart Video Panlab–Harvard Apparatus www.panlab.com secondary antibody Tracking System, Version Streptavidin–HRP Perkin Elmer Ref. NEL750001EA 2.5 https://www.perkinelmer.com/es/ Image-J 1.43 Image-J https://imagej.nih.gov/ij/ 3,3’- Vector Laboratories Ref. SK-4100 BioRender BioRender www.biorender.com Diaminabenzidine www.vectorlabs.com (DAB) Pertex mounting Medite GmbH Ref. 41-4011-00 medium www.medite.de/en/ Solution preparations 11. Cut a 7 cm copper wire. Leave between 1 and 2 cm of Tris-Buffered Saline (TBS) 10× ph.7.6 (Stock solution) copper wire and use the rest of it to roll it around the wire 60.6 gr TRIS Serva + 88 gr. NaCl in 1 L Milli-Q water of the screw twice. (Contiunued) 12. Cut the top extreme. Frontiers in Behavioral Neuroscience 08 frontiersin.org Vila-Solés et al. 10.3389/fnbeh.2022.1046259 FIGURE 6 Preparation and end result of the stimulation electrode. (A) Placement of the copper wire around the first horn, covering it until the top. (B) Placement of the solder flux paste around the wire to optimize the soldering process. Note that in this picture the excess wire in the bottom part is already cut. (C) End result of the first horn once it is soldered. (D) 1.3 mm electrode bit cut. (E) Placement of the electrode wire on the second horn. Note that once one of the extremes is stripped of insulating material, this end must face downward when placing it on the horn. (F) Placement of the resulting device in a male pin to check its connectivity. (G) Current verification of the electrode wire and copper wire with a power supply and a solution of water with dissolved NaCl. (H) Frontal view of the resulting stimulation electrode. The brown lines represent the copper wire while the gold line represents the electrode wire. Note that the soldering tin is not shown. Not to scale. (I) Frontal-lateral view of the stimulation electrode. (J) Top view of the stimulation electrode. (K) Bottom-lateral view of the stimulation electrode. (L) End result of the ground screw. Lateral view of the ground screw. The brown line represents the copper wire. (M) End result of the ground screw. Top view of the ground screw. Not to scale. Frontiers in Behavioral Neuroscience 09 frontiersin.org Vila-Solés et al. 10.3389/fnbeh.2022.1046259 13. Use the brush to collect some solder flux paste and cover incision with new cuts. Instead, a clear incision should the copper wire. be done at once. 14. Take a drop of tin with the help of the soldering iron and 8. Use a spatula to remove the periosteum membrane toward cover the copper wire. It is important to check if the copper the laterals and expose the skull. wire ends up fully stuck. If the copper wire can still be 9. Use four mosquito forceps to set aside the tissue and expose moved around the screw, repeat this process until it is fully the skull, making sure the lateral sutures, as well as Bregma attached to the screw. and Lambda are visible and accessible. 15. Once ground screws are prepared, keep them in a sealed 10. Clean the surface of the skull with hydrogen peroxide. box until used (see Figures 6L,M for the final result of the 11. Place the electrode in the lateral arm. Check that the ground screw). electrode is straight and that the tip is not bent. 12. Place the tip of the electrode on the edge of one of the Pause point: No more than 4 days should pass between lateral cranial sutures. Mark zero at the horizontal axis (Y) electrode and ground screw preparation and usage. in the digital marker. Place the tip to the opposite lateral Check Desai et al. (2014) for other ways of solving rotational suture. Divide the number obtained in two and place the movement issues. tip of the electrode in that coordinate. 13. Place the tip of the electrode on Bregma and mark zero in all coordinates of the digital marker (X, Y, Z). Stereotaxic surgery 14. Find Lambda and check the dorsoventral coordinate. If the difference is higher than 0.3 from Bregma, the inclination Timing: 1 day (30 min/rat). of the head should be readjusted. The purpose of this step is to unilaterally implant a 15. Find the coordinates AP –2.3, ML –2 and mark them on chronic electrode in the Medial Forebrain Bundle at the Lateral the skull using a pencil. Remove the arm from the working Hypothalamus. Stereotaxic coordinates in relation to Bregma area to avoid bending the electrode tip. are: –2.3 mm anterior-posterior, 2 mm medial-lateral, and 16. Apply two saline drops to the skull and drill a hole in the 8.8 mm dorsal-ventral, according to the rat brain atlas (Paxinos coordinate marked before. and Watson, 2007). All experiments below are repeated for each 17. Make two other holes anterior to the frontal sutures rat. For our experiments, we use 3-months-old male Wistar rats and two other holes anterior to posterior sutures (see from our breeding stock. Figure 7A). 18. Attach the screws to the skull. Important! The ground 1. Turn on a heating pad and place it in the surgery area. screw should be attached in the hole located in Cover the working space with paper. Turn on the lights the contralateral hemisphere for the electrode and and the digital marker. anterior to Lambda. 2. Induce anesthesia placing the animal in the anesthesia 19. Break the meninges in the electrode hole using the bent induction chamber with 5% isoflurane for 3–4 min. Check tip of the 21G needle and introduce the electrode in the breathing modifications to identify when the animal is hole until –8.8 mm DV. In case of excessive bleeding, use fully anesthetized. Afterward, remove the animal from the Cutanplast to press down on the area until bleeding stops. R chamber and shave the scalp fur with a trimmer, and put 20. Use the soldering iron and tin wire to solder the copper artificial drops over the eyes. wires from the electrode base and the ground screw 3. Place the animal in the stereotaxic apparatus placing the together. Cut any excess wire (see Figure 7A). bar inside the ear canal. Check if the head is immobile and 21. Use the solid resin and the liquid resin to create a honey- symmetry between both ears and eyes. Place the animal’s like consistency cement. Slowly let it drip over the skull, upper teeth in the nose clamp and adjust the gas mask making sure all the screws and half the base of the electrode around its nose. are covered. The cement is dry when tapping it with a 4. Reduce the isoflurane rate to 2.5%. Note that each animal spatula produces a slightly metallic sound. Caution! As may need different concentrations to maintain anesthesia. it dries, the cement should be carefully molded using a Breathing frequency should be checked during the whole spatula to avoid the formation of sharp edges that could procedure in order to assess the adequacy of the anesthesia. damage the skin. Do not push the electrode once it has 5. Administer lidocaine on the scalp and iodine with help been implanted and before the cement cures. of a cotton swab. 22. Perform one anterior and one posterior suture from the 6. Administer 2 mg/kg of analgesic electrode implant. If there is a part of the skull exposed, (Meloxicam) subcutaneously. perform more sutures where needed. The base of the 7. Take the scalpel and make an anteroposterior 1.5– electrode should be exposed, as shown in Figure 7B. 2 cm incision. It is important to avoid going over the 23. Clean the wound with iodine using a cotton swab. Frontiers in Behavioral Neuroscience 10 frontiersin.org Vila-Solés et al. 10.3389/fnbeh.2022.1046259 FIGURE 7 (A) Electrode implantation. In red, electrode implantation position in the rat skull. Note in dark gray the four screws implanted in the skull to improve the adhesion of the implant. The ground screw is implanted in the contralateral hemisphere regarding the electrode and near Lambda. (B) Rat frontal view with the electrode implanted. 24. Withdraw the animal from the stereotaxic apparatus and chewing demonstrated by gnawing noise, an increase in place it in its homecage. The homecage should remain over piloerection, and a general behavioral activation). a heating mat until the animal awakes. Place some pellets 6. Once the animal can self-administer the stimulation by inside the cage to facilitate food intake during the following itself for a minute, start a stopwatch. Stop it 5 min later. hours. 7. Write down the number of reinforcements and responses for the shaping portion. Pause point: There should a minimum of 7-days post- recovery period. All animals should be supervised daily Pause point: After establishing ICSS behavior rats must following the animal welfare protocol. All animals that have rest for a minimum of 30 min in order to return to a basal successfully recovered from the intervention may be included activation level. Session OIS-1 can be done immediately after in the behavioral procedures. resting or 24 h after the behavior shaping session. • Optimal intensity search 1 (OIS-1) Behavior shaping and optimal intensity search (OIS) Timing: 15–30 min performance dependent. • Behavior shaping 1. Restart the counter for reinforcements and responses. 2. Reduce the intensity used during the last portion of the Timing: 20–60 min (performance dependent). shaping process by 50%. These steps must be performed for both types of 3. Start a stopwatch for 2 min. After 2 min, write down the administration: self-administered (ICSS) and experimenter- number of responses performed at that intensity. administered (EAS). 4. Increase intensity by 5 µA. Start a stopwatch for 2 min. After 2 min, write down the number of responses. 1. Turn on the stimulator and select a low intensity (for 5. Repeat this process until the number of responses example, 40 µ A). decreases, for at least two consecutive intensities. If an 2. Take the animal (a cloth can be used to restrain it) animal stops responding to a specific intensity for 2 min, and connect the male connector to the female connector 10 trains of stimulation may be administered manually implanted in its skull. using the button. 3. Place the subject inside the Skinner box. The animal will 6. The current intensity at which the subject performs the start exploring the chamber spontaneously. Stimulation maximum number of responses is considered its optimal should not be administered during this initial phase of intensity 1 (OI1). exploration (Figure 8A). 4. Press the stimulation button every time the animal Caution! The number of reinforcements recorded and the approaches the wall in which the lever is located, as shown number of responses performed by the animal do not usually in Figure 8B. coincide. Each non-continuous current pulse lasts 0.2 or 0.3 s 5. The intensity can be increased gradually until the animal depending on how it is programmed. In this time interval, the shows peripheral arousal (manifested by an increase in rat can press the lever more than once. Responses are used to Frontiers in Behavioral Neuroscience 11 frontiersin.org Vila-Solés et al. 10.3389/fnbeh.2022.1046259 FIGURE 8 Intracranial electrical self-stimulation (ICSS) behavior shaping and representation of the two forms of treatment administration (self-administered and experimenter-administered). (A) Examples of the animal’s positions in which no manual stimulation should be administered. (B) Position of the rat in which manual stimulation should be administered in order to make the animal approach the lever. Please note that the electrode connections are not shown. (C) Self-administration of the treatment (ICSS). (D) Experimenter-administered treatment (EAS). The same apparatus is used for ICSS and EAS, but the lever must be hidden for the EAS treatment. calculate the rate/unit of time, which determines the optimal administration match the behavioral training sessions, but if intensity to be applied in the post-training treatment, while ICSS or EAS are designed to modulate other learning phases, the number of reinforcements indicates how much stimulation these two steps should be moved in the timeline, which may (trains) the animal has received. result in an overlap with other behavioral procedures. Pause point: A second OIS session 24 h after OIS-1 is recommended because optimal intensity usually decreases between these two sessions. Morris Water Maze acquisition Timing: 15–25 min/rat performance dependent. • Optimal intensity search 2 (OIS-2) Before the acquisition sessions take place, one habituation session is performed in order to reduce animals’ emotional Timing: 15–30 min (performance dependent). reactivity. The habituation consists in placing the animals in the pool for 60 s with no cues or platform. Once animals are 1. Follow steps in OIS-1. The current intensity in which the removed from the pool, a cued test is performed in order to subject performs the maximum number of responses is check animals’ visual acuity and motivation to find the platform. considered optimal intensity 2 (OI2). For the cued test, the platform is located in the SE quadrant. 2. IMPORTANT! The mean of OI1 and OI2 is considered the A ball with striped black and white lines is attached to the optimal intensity intensity for each subject to administer platform, ending up 20 cm above the platform surface. The cued the post-training treatment. trials consist in placing the animals in the pool to swim for a maximum of 90 s. If animals do not approach the platform Pause point: The last OIS session should ideally be during the trial, they should be manually guided to it and left performed at least 72 h before the MWM training. Shorter there for 15 s. Habituation and cued tests were performed 3 days intervals can have an anterograde effect on the performance before the acquisition sessions. of the task, potentially masking the effects of the post-training The acquisition sessions consist of five consecutive days with ICSS/EAS treatment. In this protocol, the days of the treatment six trials of 90 s each. In each trial, subjects are introduced Frontiers in Behavioral Neuroscience 12 frontiersin.org Vila-Solés et al. 10.3389/fnbeh.2022.1046259 into the pool from different cardinal points (N, E, W, S). 2. A total of 2,500 trains of stimulation should be Platform is always located in the SE quadrant. The main variable administered, aiming to deliver each train 0.5–1 s apart. assessed was the Escape latency, which refers to the time that 3. Remove the animal and return it to its home cage. subjects take to find the platform. As main control variables, we 4. Write down the total duration of the treatment, and any measured the Time spent near walls, the swimming Speed, and relevant observations. the Total distance the animals swim. The time spent near walls measures thigmotaxis, which is an indicator of anxiety (Treit and Fundytus, 1988; Huang et al., 2012). • Non-stimulated animals Pause point: Since we perform post-training stimulation, there is not an actual pause between the behavioral training and the treatment administration. The only time delay is due to the 1. Place the animal inside the Skinner box. rat withdrawal from the MWM and properly drying the subject 2. During ICSS shaping and optimal intensity establishment, with towels before continuing with the protocol. leave animals for 20 min in the Skinner box. 3. During treatment sessions, leave them in the Skinner box for 45 min. Intracranial electrical stimulation 4. Remove the animal and place it in its homecage. treatment Pause point: This pause point depends on the timeline Timing: 30–60 min/rat (performance dependent). and the main objectives of the experiment. In this study, IMPORTANT! For all conditions, place a heating pad animals underwent a retention test 72 h after the last post- behind the Skinner box. Especially if animals have just training treatment. performed the Morris Water Maze, in which water temperature is around 70–74◦ F (21–23◦ C). • Self-administration Morris Water Maze retention Self-administration treatment is depicted in Figure 8C. Timing: 3 min/rat. Seventy-two hours after the last acquisition session, animals 1. Turn on the stimulator and select the chosen intensity (OI) performed the retention test, which consisted in removing the for that subject. hidden platform from the pool and placing the animal in the 2. Take the animal (a cloth can be used to restrain it) and pool for 60 s starting from the East. Variables assessed for the connect the male connector to the female connector of the retention test were analyzed for the totality of the trial duration electrode implanted in its skull. (60 s), and the first half of the trial (30 s), given that the accuracy 3. Place the subject inside the Skinner box. and level of performance could change throughout the retention 4. Once the animal is facing the wall with the lever, press the test in the MWM (Blokland et al., 2004). The variables analyzed button to administer electrical stimulation. were: Percentage of time spent in the target quadrant in the first 5. The subject should immediately start self-administering half of the trial (Q30) and its totality (Q60); Average distance the treatment. Start a stopwatch when this happens. to the target (DT30 and DT60), Percentage of time spent in 6. Leave the animal self-administer 2,500 trains the target annulus (Ann30 and Ann60), Time spent near walls of stimulations. (W30 and W60) and Wishaw’s Error (WE), which measures the 7. Once the subject reaches 2,500 trains (reinforcements), time they animals spent in a 30 cm wide corridor starting at the turn off the stimulator. East entry point and the platform and indicates accuracy of the 8. Remove the animal and return it to its home cage. trajectory. 9. Write down the total duration of the treatment and Pause point: Subjects were euthanized 90 min after the total responses. retention test took place. • Experimenter-administration Tissue collection and immunohistochemical study Experimenter-administration treatment is depicted in Figure 8D. Timing: 2 days for IHC. This step of the protocol aims to assess neuronal activity 1. Follow steps 1–3 of the self-administration protocol above. differences between groups after the retention session. Frontiers in Behavioral Neuroscience 13 frontiersin.org Vila-Solés et al. 10.3389/fnbeh.2022.1046259 Ninety minutes after the retention test ended, the animals Results received a pentobarbital overdose (150 mg/Kg, i.p.) and were transcardially perfused with a solution of 0.1M of phosphate Quantification and statistical analysis buffer saline (PBS), pH 7.4, followed by a solution of 4% paraformaldehyde in PBS. Brains were removed and post- Statistical analyses are performed using SPSS v23 (SPSS Inc., fixed in 4% paraformaldehyde in PBS, then cryoprotected Chicago, IL, USA). A total of 21 Wistar albino male rats (Rattus in 15% and 30% sucrose in PBS and stored at –80◦ C. Norvegicus) were used in this experiment with a mean of age of Serial coronal sections (30 um) were obtained in a cryostat 113 days (SD = 4.00) and a mean weight of 389.57 g (SD = 7.00) (Cryocut 1800, with 2020 JUNG microtome) at –20◦ C at the beginning of the experiment. Four animals were excluded and stored at –80◦ C until immunostaining. Free-floating from the experiment. Three subjects were excluded because coronal sections were incubated with 0.3% H2O2 in Tris- they were extreme outliers in behavioral tasks (>2.0 SD points buffered saline (TBS) for 30 min and in 1% Bovine from mean in more than two acquisition sessions). One subject Serum Albumin (BSA) in 0.3% Triton X-100 in TBS was excluded because of recording problems issues during (TBS-T) for 30 min. Sections were incubated in primary behavioral tests. No animals were excluded because of electrode antibody mouse anticFos (Santa Cruz Biotechnology Inc., misplacing, as shown in Figure 9. For behavioral and molecular Santa Cruz, CA, USA; sc-166940) at 1:500 in 0.1% BSA analysis, the total sample consisted of 17 subjects (ICSS: n = 6; in TBS-T overnight at 4◦ C. The next day samples were EAS: n = 6; SHAM: n = 5). All subjects were kept in an ad libitum incubated with biotinylated goat anti-mouse-IgG secondary regime of food and water. antibody (Jackson Immunoresearch Inc., Ely, United Kingdom; Analysis of the performance in the MWM is conducted ref.: 115-065-166) at 1:500 in 0.1% BSA in TBS-T for using a 3 × 4 mixed analysis of variance (GROUP × SESSION) 60 min at room temperature and subsequently treated with for the acquisition phase. An independent sample t-test analysis Streptavidin-HRP (AKOYA Biosciences, Marlborough, MA, was used for the first acquisition session (S1) and the 72-h United States) at 1:2500 in TBS-T for 120 min, followed by retention test. Additionally, a t-test was used to compare the treatment with 3,30 -Diaminobenzidine (DAB) solution (Vector time animals spent in the target quadrant during the retention Laboratories, Newark, NJ, United States) for 5 min. Finally, test to the chance level (25%). sections were mounted onto slides, dehydrated, and coverslips To analyze c-Fos quantitative data we use a 3 × 2 mixed were placed with Pertex mounting medium (Medite GmbH, analysis of variance (GROUP × HEMISPHERE) for each brain Wollenweberstraße, Germany). Negative controls without area studied. When HEMISPHERE factor was not significant, primary antibodies were included. the mean of the ipsilateral and contralateral hemisphere was FIGURE 9 Electrode tip location for the intracranial electrical self-stimulation (ICSS) and experimenter-administered stimulation (EAS) groups. ICSS group is represented by an orange triangle and the EAS group is represented by a blue box. MFB, medial forebrain bundle; f, fornix; PAV, paraventricular hypothalamic nucleus; AHP, anterior hipotalamic area; 3V, third ventricle; opt, optic tract; EP, entopeduncular nucleus; VMH, ventromedial hypothalamus. Frontiers in Behavioral Neuroscience 14 frontiersin.org Vila-Solés et al. 10.3389/fnbeh.2022.1046259 analyzed. Microphotographs of the specific brain regions are (F 1 ,14 = 77.229; P < 0.001), with some inflection (quadratic taken with a 10 × objective lens using a BX-41 Olympus function: F 1 ,14 = 5.431; P = 0.035) possibly due to the microscope attached to an Olympus DP-70 digital camera floor effect that is usually observed in the last sessions. (Japan). Appropriate gray threshold and particle size are set Despite the fact that all the groups showed an improvement for each area and maintained for all subjects. The image in the execution throughout the sessions, the two groups analysis software Image-J 1.431 is employed to bilaterally that received treatment showed lower escape latencies in count the number of c-Fos immunostained nuclei using comparison with the non-stimulated group (ICSS vs. non- regions of interest (ROIs) (see Figures 10E–H in the Section stimulated: P = 0.003; EAS vs. non-stimulated: P = 0.001). “Results”). Three histological sections are counted and averaged Thus, both forms of administration equally facilitate the for all areas. In order to remove background noise, each acquisition of the task. These effects start immediately after image is digitally smoothed and subtracted from the original. the first treatment session, and are maintained throughout See Figures 10I–L for representative immunohistochemistry the acquisition phase. No differences in control variables were outcomes. observed. Retention Optimal intensity of ICSS and Compared to chance level (25%) during the first half of the responses rate evolution test (Q30), both groups that received stimulation spent more time in the target quadrant (ICSS: t 5 = 3.306, P = 0.021; EAS: The following figures depict representative optimal t 5 = 5.469, P = 0.003), while the non-stimulated group did intensities and response rate obtained using standard ICSS not (t 4 = 1.824, P = 0.142). However, when we look at the parameters. Regarding OI, a decline is generally observed whole test (Q60), all of the groups spent more time in the between OI1 and OI2 sessions, which stabilizes if more sessions target quadrant compared to chance level (ICSS: t 5 = 3.785, are carried out. In this experiment, only OI1 and OI2 sessions P = 0.013; EAS: t 5 = 5.359, P = 0.003; non-stimulated: t 4 = 3.391, were performed. However, the number of OIS sessions will P = 0.027), as shown in Figure 12B. Regarding the time depend on the purpose of the study (Figure 11A). spent near the walls during the first half of the retention The response curve at different intensities usually fits an test (W30), significant differences between groups were found inverted-U (Figure 11B). The intensity that induces the highest (F 2 ,16 = 4.175, P = 0.038). Specifically, both stimulated groups response rate is called the optimal intensity. This figure also spent less time near the walls compared with the non-stimulated shows the intensity threshold of ICSS. group (Helmert contrast: P = 0.021), as shown in Figure 12C. There are no differences between groups in any other variables analyzed. Learning and memory outcomes Acquisition Immunohistochemistry outcomes The main variable used to assess performance during the acquisition of the MWM is the escape latency, or the time We measured c-Fos expression after retention at 72 h subjects spend trying to find the hidden platform. No significant to determine neuronal activity in the primary motor cortex differences among groups in the first session (S1) were found (M1), nucleus accumbens core (NAcC), centromedial thalamic (P = 0.199), which indicates that all groups began the acquisition nucleus (CMT), and hippocampal CA1 region. A mixed 3 × 2 phase at comparable levels of performance. These results allow ANOVA (GROUP × HEMISPHERE) was used. us to rule out any effects of the previous stimulation that Regarding the M1 (Figure 10A), bilateral differences ICSS/EAS subjects had received before MWM training. were observed among groups (F 2 ,12 = 6.309, P = 0.013). A repeated measures ANOVA analysis A post hoc (Tukey HSD) contrast showed significantly higher (GROUP × SESSION) was used to study the escape latencies c-Fos expression in ICSS animals compared to the groups that for the rest of the acquisition sessions (S2–S5). GROUP did not perform the lever-press response (EAS, P = 0.008; non- (F 2 ,14 = 10.098, P = 0.002) and SESSION (F 3 ,42 = 29.174, stimulated, P = 0.012). Similar bilateral results were observed in P < 0.001) factors were resulted significant, but their interaction the CMT (GROUP: F 2 ,13 = 15.708, P < 0.001) (Figure 10C), was not significant (F 6 ,42 = 1.816, P = 0.119). As it can be where the ICSS group also exhibited more cFos + neurons observed in Figure 12A, the evolution of the performance than the other two groups (EAS, P = 0.005; non-stimulated, throughout sessions is mainly adjusted to a linear function P < 0.0001). In the NAcC (Figure 10B), differences among groups were hemisphere-dependent (GROUP × HEMISPHERE: 1 http://rsb.info.nih.gov/ij/ F 2 ,12 = 3.944, P = 0.048). In the ipsilateral hemisphere, the Frontiers in Behavioral Neuroscience 15 frontiersin.org Vila-Solés et al. 10.3389/fnbeh.2022.1046259 FIGURE 10 Effects of different administrations (self-administered or experimenter-administered) on c-Fos expression in the primary motor cortex, nucleus accumbens core, centromedial thalamus, and CA1. (A–D) c-Fos + neurons/mm2 (±SE) in the (A) primary motor cortex (M1), (B) NAcC, (C) CMT, and (D) CA1. The ipsilateral hemisphere corresponds to the right hemisphere, in which the electrode was implanted. Panels (A,C) show the mean expression of c-Fos of both hemispheres. (E–H) Regions of interest (ROI) for the quantification of c-Fos + neurons of the (E) M1, (F) nucleus accumbens core (NAcC), (G) centromedial thalamus, and (H) the cornus ammonis 1 (CA1), with their corresponding coordinates regarding Bregma. Images adapted from Paxinos and Watson (2007). In regions in which HEMISPHERE factor was not significant, the mean of both hemispheres is represented (A,C). Significant differences are shown as *P ≤ 0.05, **P ≤ 0.01. (I–L) Representative images of c-Fos expression for each group and area studied taking into account the ipsilateral and contralateral hemispheres. ***P < 0.001. ICSS condition caused a greater level of c-Fos expression non-stimulated groups. Due to issues in the immunolabeling than the non-stimulation condition (P = 0.025), while of the samples, the results of the contralateral hemisphere of the EAS group did not differ from either the ICSS or the the CA1 region were not reliable. Based on previous work Frontiers in Behavioral Neuroscience 16 frontiersin.org Vila-Solés et al. 10.3389/fnbeh.2022.1046259 FIGURE 11 Intracranial electrical self-stimulation (ICSS) variables evolution. (A) Optimal intensity evolution throughout the shaping session and the optimal intensity establishment sessions. (B) Response rate depends on the intensity (µA) used. showing that ICSS causes similar bilateral activation of CA1 consistently shown to be the most efficient (Segura-Torres et al., (Huguet et al., 2009), only ipsilateral CA1 has been included 1988; Redolar-Ripoll et al., 2002; Soriano-Mas et al., 2007). in the analysis. No differences between groups were observed Thus, this protocol describes how to perform post-training (Figure 10D). administration of the treatment, be it ICSS or EAS. Finally, another limitation is that the assessment of neuronal activation by c-Fos is time-sensitive (Chaudhuri et al., 2000). Thus, we are only able to link neuronal activation to the Advantages and limitations performance in the retention test, as that is the task the animals undergo 90 min before euthanasia and tissue extraction. The main advantage of ICSS is the relative ease with which experimental animals acquire this behavior, which allows for the physiological stimulation of the BRS. In addition, Troubleshooting it is proven to not cause dependency or addiction, so it does not involve tolerance phenomena nor resistance to Problem 1: The electrode implant falls down. extinction. Furthermore, post-mortem histological control of Potential solution: the location of the electrode is not necessary because the successful ICSS behavior itself is a guarantee of accurate (1) Avoid excessive manipulation of the electrode. placement. Finally, the target area is relatively large and (2) Tighten the screws deeper into the skull. after enough training in stereotaxic surgery, the percentage (3) Before applying the acrylic cement to the skull, dry the of error for accurate implantation is less than 10%. It surface completely. should be noted that ICSS is considered to be innocuous and can be obtained in different species, both in male and Problem 2: An animal does not show ICSS behavior. female subjects. Potential solution: One possible limitation regarding this protocol is the time of treatment administration. Depending on experimental (1) We can remove the animal from the Skinner box and check objectives, the stimulation treatment could be administered all connections between the stimulator, the rat electrode at different times in relation to the learning task or other device, and the lever. To check that all the components relevant procedures. For instance, pre-training treatment of the circuit work correctly, you may use a resistor. It is (before training) would affect the acquisition of the task, while advisable to have several spare cables available. pre-retention (before the probe test) would affect recall, and (2) Although not very common, some animals seem to receive post-training (after training) would affect the consolidation insufficient current intensity on the first day of shaping process. The treatment can also be administered non- behavior. This may be due to something that increases contingently to the learning process, in between phases of the tissue resistance, such as a blood clot. This is usually training. All of these different designs have been experimentally resolved without requiring any type of intervention, so it tested for ICSS, and the post-training administration has is recommended to test the animal again at a later time. Frontiers in Behavioral Neuroscience 17 frontiersin.org Vila-Solés et al. 10.3389/fnbeh.2022.1046259 FIGURE 12 Effects of administration type (self-administered or experimenter-administered) on the acquisition and retention of a spatial task in the Morris Water Maze (MWM). (A) Mean escape latencies (±SE) of intracranial electrical self-stimulation (ICSS), experimenter-administered stimulation (EAS), and non-stimulated groups for the five sessions of the acquisition phase. Black arrow indicates the start of the treatment administration (self-administered, experimenter-administered, or non-stimualted). Both ICSS and EAS groups showed a significant statistically lower escape latencies compared to the non-stimulated group once the treatment had begun (S2–S5). **P ≤ 0.01. (B) Time spent in the target quadrant (±SE) during the retention test for the first half of the test (Q30) and the total of the test (Q60). Note that the chance level (25%) is shown in the graph as a dashed horizontal line. (C) Time spent near walls (±SE) during the retention test for the first half of the test (W30) and the total of the test (W60). Significant differences are shown as *P ≤ 0.05, **P ≤ 0.01. (3) In some cases, the rat never shows ICSS behavior. For this may continue in the experiment, using lower intensities. reason, it is necessary to periodically check the stereotaxic Seizures do not affect performance on the learning apparatus, and be very careful in the elaboration of the task, but they affect the number of c-Fos + neurons, electrodes. The moment of its implantation in the brain is especially in the dentate gyrus, thus, depending on also critical. The electrode wire should always be prevented the molecular objectives, this should be taken into from being accidentally bent or damaged. For example, consideration. once the hole in the skull is performed, perforate the meninges with a needle. If this step is forgotten, the Problem 4: Animals learn the behavioral task (in this case, electrode wire could be bent as it passes through the hard the spatial task) faster than expected, in which case the effects of rat meninges, and this could affect the path of the electrode. the treatment are not observable. Potential solution: Problem 3: Animals display abnormal behaviors, such as stereotypic behavior, extreme peripheral arousal or even (1) Make the task more difficult by reducing the number of seizures during stimulation, which may hinder its ability to self- trials per session (from 6 to 4 or to 2), or by reducing the stimulate. Increased piloerection, teeth chattering, grooming number of the total sessions performed (Chamorro-López and occasional ejaculation in males are normal occurrences. It is et al., 2015; García-Brito et al., 2018). also normal for the animal to occasionally rest for short periods during the ICSS treatment. Potential solution: Discussion (1) Lower the intensity of stimulation, if possible. This paper shares standardized guidelines for using (2) Closely monitor the animal’s behavior during ICSS or EAS rewarding MFB stimulation as a treatment to improve and pay special attention to the time it takes to complete learning and memory in rats. The materials and steps the portion of the session. If the animal continuously described in this protocol have been used to assess the disengages from the ICSS behavior or if the abnormal effects of self- vs. experimenter-administration on the behavior is too severe, it should not be included in the MWM performance, as well as on the expression of c-Fos stimulation groups. after the retention test. To our knowledge, this is the (3) Seizures: the start of a convulsive episode can be first time that ICSS and EAS have been compared in a assessed by the ceasing of self-stimulation activity spatial memory task. and slobbering. In these cases, stop the stimulation The behavioral data show that reinforcing stimulation of immediately. Wait a few minutes before removing the the MFB is an effective treatment for facilitating the acquisition animal from the cage. General parameters of the subject of a spatial learning task in rats, regardless of whether it should be checked over the following days. If seizures is self-administered or externally administered. The effects were provoked due to high intensity levels, animals of both forms of administration are evident after the first Frontiers in Behavioral Neuroscience 18 frontiersin.org Vila-Solés et al. 10.3389/fnbeh.2022.1046259 administration of the treatment, and this effect is maintained immediately after stimulation. Nevertheless, ICSS effects seem throughout the acquisition sessions. The facilitating effect of to be more long-lasting. Both groups received the same amount ICSS is in agreement with previous results in the MWM of stimulation; therefore, the most likely explanation is twofold. (Ruiz-Medina et al., 2008; Chamorro-López et al., 2015), and other types of tasks, such as active avoidance conditioning (Segura-Torres et al., 2010). It has been shown that the potency Key resources table of the effect is related to the amount of training, since non- stimulated animals are able to reach a better performance On the one hand, in the ICSS condition, the experimenter when they undergo more training (Redolar-Ripoll et al., 2002; may readjust the intensity of stimulation based on the ICSS Ruiz-Medina et al., 2008; Chamorro-López et al., 2015). This behavior. On the other hand, the rats in this condition are could potentially reduce the scope for action of the treatment. able to regulate their own level of motivation or arousal by However, despite using a higher number of trials per session adjusting the response rate, and usually showing obvious signs in the present study, both the ICSS and EAS-treated subjects of peripheral arousal and behavioral activation (Nieh et al., showed an improved performance compared to the non- 2016). Higher expression of c-Fos in the arousal-related CMT stimulated animals. This would be in agreement with results nucleus of the ICSS group could support this idea. Moreover, the showing that ICSS could be even more powerful than the ipsilaterality of the observed effect in the NAcC and bilaterality training itself (Redolar-Ripoll et al., 2002). in the CMT agrees with previous results which show greater In contrast, no differences were observed between the ipsilateral activation only in the areas most directly connected stimulated and non-stimulated animals in the variables assessed to the stimulated region (Arvanitogiannis et al., 1996). in the retention test. Does that mean that MFB stimulation has In summary, both forms of administrating rewarding had no long-term effects? When assessing the retention level stimulation facilitate a hippocampus-dependent learning compared to chance within each group in the first half of the task, although they present differences regarding long-term test, only stimulated animals spend significantly more time in activation levels in some brain regions related to arousal, and the target quadrant. This suggests that they recalled the location motor behavior. These results suggest that MFB is a promising of the platform earlier than non-stimulated animals. Moreover, target for use in DBS treatment of memory-related disorders since the spatial MWM is considered to be a hippocampus- in humans, even if the stimulation is not self-administered. dependent memory task (Vorhees and Williams, 2014a) a Moreover, they support the idea that the results obtained using study of CA1 activation was carried out. Similar c-Fos levels ICSS in an animal model are useful to guide clinical research. found in CA1 of all groups support the idea that they all end up presenting comparable retention levels, probably due to the intense training they had undergone. In fact, ICSS Data availability statement manages to produce long-term facilitating effects when animals receive fewer trials during acquisition (García-Brito et al., 2020). The raw data supporting the conclusions of this article will Interestingly, stimulated animals spent significantly less time be made available by the authors, without undue reservation. near walls in the retention test compared to non-stimulated groups. This reduction in thigmotactic behavior could be related with a better retention. On the other hand, this outcome suggests Ethics statement a potential anxiolytic effect of the MFB stimulation. Despite the fact that the behavioral outcome suggests The animal study was reviewed and approved by equivalent therapeutic potential, the results of the c-Fos Departament d’Agricultura, Ramaderia, Pesca i Alimentació, expression in M1, NAcC, and CMT, suggest possible differences Generalitat de Catalunya (DARP Ref. 7350). between both administration forms. As expected, the ICSS group showed increased activation of the primary motor cortex, which would be explained by the persistent lever- Author contributions pressing behavior required to obtain electrical stimulation (Shankaranarayana Rao et al., 1998). LV-S: investigation and writing—original draft. SG-B: Surprisingly, although both ICSS and EAS groups received investigation and writing—review and editing. LA-V: rewarding stimulation, only the ICSS group showed more conceptualization, methodology, and writing—review and activation in the NAcC compared to the non-stimulated editing. EK: conceptualization, methodology, writing—review animals. This is known to be a critical region of the mesolimbic and editing, and funding acquisition. GH: conceptualization, system (Baik, 2020). Since c-Fos was assessed 72 h after methodology, supervision, and funding acquisition. IM-B: the last treatment administration, we cannot know whether conceptualization. PS-T: conceptualization, writing—review activation of this nucleus was similar or different for both groups and editing, project administration, and funding acquisition. Frontiers in Behavioral Neuroscience 19 frontiersin.org Vila-Solés et al. 10.3389/fnbeh.2022.1046259 All authors contributed to the article and approved the that could be construed as a potential conflict of submitted version. interest. Funding Publisher’s note This work was supported by PID2020-117101RB-C21 and All claims expressed in this article are solely those of the PID2020-117101RB-C22 grants from Ministerio de Ciencia e authors and do not necessarily represent those of their affiliated Innovación (Spain). organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed Acknowledgments or endorsed by the publisher. All images were created with BioRender.com. Supplementary material Conflict of interest The Supplementary Material for this article can be found The authors declare that the research was conducted online at: https://www.frontiersin.org/articles/10.3389/fnbeh. in the absence of any commercial or financial relationships 2022.1046259/full#supplementary-material References Aldavert-Vera, L., Huguet, G., Costa-Miserachs, D., Ortiz, S. P., de, Kádár, spatial memory task. Behav. Neurosci. 129, 720–30. doi: 10.1037/bne000 E., Morgado-Bernal, I., et al. (2013). Intracranial self-stimulation facilitates 0098 active-avoidance retention and induces expression of c-Fos and Nurr1 in rat Chaudhuri, A., Zangenehpour, S., and Rahbar-Dehgan, F. Y. (2000). Molecular brain memory systems. Behav. Brain Res. 250, 46–57. doi: 10.1016/J.BBR.2013.0 maps of neural activity and quiescence. Acta Neurobiol. Exp. 60, 403–10. 4.025 Aldehri, M., Temel, Y., Alnaami, I., Jahanshahi, A., and Hescham, S. (2018). Desai, S. J., Bharne, A. P., Upadhya, M. A., Somalwar, A. R., Subhedar, N. K., and Deep brain stimulation for Alzheimer’s disease: An update. Surg. Neurol. Int. 9:58. Kokare, D. M. (2014). A simple and economical method of electrode fabrication doi: 10.4103/SNI.SNI_342_17 for brain self-stimulation in rats. J. Pharmacol. Toxicol. Methods 69, 141–9. doi: 10.1016/j.vascn.2013.12.006 Arvanitogiannis, A., Flores, C., Pfaus, J. G., and Shizgal, P. (1996). Increased ipsilateral expression of Fos following lateral hypothalamic self-stimulation. Brain Fenoy, A. J., Schulz, P. E., Selvaraj, S., Burrows, C. L., Zunta-Soares, G., Durkin, Res. 720, 148–154. doi: 10.1016/0006-8993(96)00096-0 K., et al. (2018). A longitudinal study on deep brain stimulation of the medial forebrain bundle for treatment-resistant depression. Transl. Psychiatry 8:111. doi: Baik, J. H. (2020). Stress and the dopaminergic reward system. Exp. Mol. Med. 10.1038/S41398-018-0160-4 52:1879–1890. doi: 10.1038/S12276-020-00532-4 García-Brito, S., Aldavert-Vera, L., Huguet, G., Álvarez, A., Kádár, E., and Baird, T. R., Davies, R. A., Glennon, R. A., Peace, M. R., and Negus, S. S. Segura-Torres, P. (2018). Increased training compensates for OX1R blockage- (2021). A strategy to prioritize emerginc drugs of abuse for analysis: Abuse impairment of spatial memory and c-Fos expression in different cortical and liability testing using intracranial self-stimulation (ICSS) in rats and validation subcortical areas. Behav. Brain Res. 353, 21–31. doi: 10.1016/j.bbr.2018.05.028 with α-pyrrolidinohexanophenone (α-PHP). Emerg. Trends Drugs Addict. Health 1:100004. doi: 10.1016/j.etdah.2021.100004.A García-Brito, S., Aldavert-Vera, L., Huguet, G., Kádár, E., and Segura-Torres, P. (2020). Orexin-1 receptor blockade differentially affects spatial and visual Bamford, N. S., Wightman, R. M., and Sulzer, D. (2018). Dopamine’s effects discrimination memory facilitation by intracranial self-stimulation. Neurobiol. on corticostriatal synapses during reward-based behaviors. Neuron 97:494–510. Learn. Mem. 169:107188. doi: 10.1016/j.nlm.2020.107188 doi: 10.1016/J.NEURON.2018.01.006 García-Brito, S., Morgado-Bernal, I., Biosca-Simon, N., and Segura-Torres, Bennett, S., and Thomas, A. J. (2014). Depression and dementia: Cause, P. (2017). Intracranial self-stimulation also facilitates learning in a visual consequence or coincidence? Maturitas 79:184–90. doi: 10.1016/J.MATURITAS. discrimination task in the Morris water maze in rats. Behav. Brain Res. 317, 2014.05.009 360–366. doi: 10.1016/J.BBR.2016.09.069 Bewernick, B. H., Kayser, S., Gippert, S. M., Switala, C., Coenen, V. A., and Huang, Y., Zhou, W., and Zhang, Y. (2012). Bright lighting conditions during Schlaepfer, T. E. (2017). Deep brain stimulation to the medial forebrain bundle for testing increase thigmotaxis and impair water maze performance in BALB/c mice. depression- long-term outcomes and a novel data analysis strategy. Brain Stimul. Behav. Brain Res. 226, 26–31. doi: 10.1016/J.BBR.2011.08.043 10:664–671. doi: 10.1016/J.BRS.2017.01.581 Huguet, G., Aldavert-Vera, L., Kádár, E., de Ortiz, S., Morgado-Bernal, Blokland, A., Geraerts, E., and Been, M. (2004). A detailed analysis of rats’ I., and Segura-Torres, P. (2009). Intracranial self-stimulation to the lateral spatial memory in a probe trial of a morris task. Behav. Brain Res. 154, 71–5. hypothalamus, a memory improving treatment, results in hippocampal changes doi: 10.1016/j.bbr.2004.01.022 in gene expression. Neuroscience 162, 359–74. doi: 10.1016/J.NEUROSCIENCE. Carlezon, W. A., and Chartoff, E. H. (2007). Intracranial self-stimulation (ICSS) 2009.04.074 in rodents to study the neurobiology of motivation. Nat. Protoc. 2, 2987–95. Huguet, G., Kádár, E., Serrano, N., Tapias-Espinosa, C., García-Brito, S., doi: 10.1038/nprot.2007.441 Morgado-Bernal, I., et al. (2020). Rewarding deep brain stimulation at the medial Chamorro-López, J., Migúns, M., Morgado-Bernal, I., Kastanauskaite, A., forebrain bundle favours avoidance conditioned response in a remote memory Selvas, A., Cabané-Cucurella, A., et al. (2015). Structural plasticity in hippocampal test, hinders extinction and increases neurogenesis. Behav. Brain Res. 378:112308. cells related to the facilitative effect of intracranial self-stimulation on a doi: 10.1016/j.bbr.2019.112308 Frontiers in Behavioral Neuroscience 20 frontiersin.org Vila-Solés et al. 10.3389/fnbeh.2022.1046259 Kádár, E., Ramoneda, M., Aldavert-Vera, L., Huguet, G., Morgado-Bernal, I., consolidation in rats with little training. Neurobiol. Learn. Mem. 89, 574–81. and Segura-Torres, P. (2014). Rewarding brain stimulation reverses the disruptive doi: 10.1016/j.nlm.2007.11.005 effect of amygdala damage on emotional learning. Behav. Brain Res. 274, 43–52. Segura-Torres, P., Aldavert-Vera, L., Gatell-Segura, A., Redolar-Ripoll, D., and doi: 10.1016/J.BBR.2014.07.050 Morgado-Bernal, I. (2010). Intracranial self-stimulation recovers learning and Lyons, M. K. (2011). Deep brain stimulation: Current and future clinical memory capacity in basolateral amygdala-damaged rats. Neurobiol. Learn. Mem. applications. Mayo Clin. Proc. 86, 662–72. doi: 10.4065/mcp.2011.0045 93, 117–26. doi: 10.1016/j.nlm.2009.09.001 Milner, P. M. (1991). Brain-stimulation reward: A review. Can. J. Psychol. 45, Segura-Torres, P., Capdevila-Ortís, L., Martí-Nicolovius, M., and Morgado- 1–36. doi: 10.1037/h0084275 Bernal, I. (1988). Improvement of shuttle-box learning with pre- and post-trial intracranial self-stimulation in rats. Behav. Brain Res. 29, 111–117. doi: 10.1016/ Morgado-Bernal, I., and Segura-Torres, P. (2022). Intracranial self-stimulation 0166-4328(88)90058-7 and memory in rats : A sistematic review. Psicothema 34, 446–453. doi: 10.7334/ psicothema2022.66 Shankaranarayana Rao, B. S., Raju, T. R., and Meti, B. L. (1998). Long-lasting Nieh, E. H., Vander Weele, C. M., Matthews, G. A., Presbrey, K. N., Wichmann, structural changes in CA3 hippocampal and layer V motor cortical pyramidal R., Leppla, C. A., et al. (2016). Inhibitory input from the lateral hypothalamus to neurons associated with self-stimulation rewarding experience: A quantitative the ventral tegmental area disinhibits dopamine neurons and promotes behavioral golgi study. Brain Res. Bull. 47, 95–101. doi: 10.1016/S0361-9230(98)00056-2 activation. Neuron 90, 1286–1298. doi: 10.1016/J.NEURON.2016.04.035 Soriano-Mas, C., Redolar-Ripoll, D., Guillazo-Blanch, G., Morgado-Bernal, Olds, J., and Milner, P. (1954). Positive reinforcement produced by electrical I., and Segura-Torres, P. (2007). Intracranial self-stimulation after memory stimulation of septal area and other regions of rat brain. J. Comp. Physiol. Psychol. reactivation: Immediate and late effects. Brain Res. Bull. 74, 51–57. doi: 10.1016/j. 47, 419–27. doi: 10.1037/h0058775 brainresbull.2007.05.003 Paxinos, C., and Watson, G. (2007). The Rat Brain in Stereotaxic Coordinates, Treit, D., and Fundytus, M. (1988). Thigmotaxis as a test for anxiolytic activity in 6th Edn. Cambridge, MA: Elsevier Academic Press. rats. Pharmacol. Biochem. Behav. 31, 959–62. doi: 10.1016/0091-3057(88)90413-3 Puig-Parnau, I., Garcia-Brito, S., Faghihi, N., Gubern, C., Aldavert-Vera, L., Vorhees, C. V., and Williams, M. T. (2014b). Value of water mazes for Segura-Torres, P., et al. (2020). Intracranial self-stimulation modulates levels assessing spatial and egocentric learning and memory in rodent basic research and of SIRT1 protein and neural plasticity-related microRNAs. Mol. Neurobiol. 57, regulatory studies. Neurotoxicol. Teratol. 45, 75–90. doi: 10.1016/J.NTT.2014.07. 2551–2562. doi: 10.1007/s12035-020-01901-w 003 Redolar-Ripoll, D., Aldavert-Vera, L., Soriano-Mas, C., Segura-Torres, P., Vorhees, C. V., and Williams, M. T. (2014a). Assessing spatial learning and and Morgado-Bernal, I. (2002). Intracranial self-stimulation facilitates memory memory in rodents. ILAR J. 55, 310–32. doi: 10.1093/ilar/ilu013 consolidation, but not retrieval: Its effects are more effective than increased training. Behav. Brain Res. 129, 65–75. doi: 10.1016/S0166-4328(01)00325-4 Wei, G., Irish, M., Hodges, J. R., Piguet, O., and Kumfor, F. (2020). Disease-specific profiles of apathy in Alzheimer’s disease and behavioural-variant Ruiz-Medina, J., Redolar-Ripoll, D., Morgado-Bernal, I., Aldavert-Vera, L., frontotemporal dementia differ across the disease course. J. Neurol. 267, 1086– and Segura-Torres, P. (2008). Intracranial self-stimulation improves memory 1096. doi: 10.1007/S00415-019-09679-1 Frontiers in Behavioral Neuroscience 21 frontiersin.org