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Doctoral Thesis DOCTORAL THESIS 2019 A multiparametric study of the psychobiological mechanisms operating in the incubation of seeking of drugs and natural reinforcers in the laboratory rat Estudio multiparamétrico de los mecanismos psicobiológicos de la incubación de la búsqueda de drogas y reforzadores naturales en ratas de laboratorio DAVID ROURA MARTÍNEZ Doctoral Program in Psychology of Health Thesis advisors: Dr. ALEJANDRO HIGUERA MATAS Dr. EMILIO AMBROSIO FLORES RESEARCH FUNDED BY: Spanish Ministry of Economy and Competitiveness: SAF2013-47520-P PSI2016-80541-P PSI2010-20355 Spanish Ministry of Health, Social Services and Equality: - Red de Trastornos Adictivos: RTA-RD12/028/0020 of the Institute of Health Carlos III - Plan Nacional Sobre Drogas: 2012I057 General Directorate of Research of the Community of Madrid: S-2011/BMD-2308, Program of I+D+I Activities CANNAB-CM Universidad Nacional de Educación a Distancia: Plan for the Promotion of Research Predoctoral fellowship European Union: JUST/2013/DPIP/AG/4823-EU MADNESS AGRADECIMIENTOS Estoy profundamente agradecido a mis directores de tesis, Alejandro y Emilio, por la confianza que desde el principio han depositado en mí y por la oportunidad que me dieron de trabajar en su grupo de inves- tigación. Su profesionalidad y calidad humana son encomiables. Así mismo, quiero agradecer a Arturo y María Rosa por confiar en mí y escogerme para investigar con ellos durante el último año y medio de mi estancia en Madrid. Sin lugar a dudas, aquel puesto fue tan oportuno como provechoso. A continuación citaré, cronológicamente, a aquellas personas con las que he compartido parte de mi tra- yectoria en forma de compañeros, mentores, amigos y/o enlaces. Cuando cite a varios lo haré en orden alfabético. La primera persona a quien quiero agradecer es mi madre. Ella me animó a que me mudara, habiendo cumplido la mayoría de edad, de mi pueblo, Pasajes (Gipuzkoa), a Bilbao (Bizkaia), a estudiar aquello que quería, Biología, ya que en el campus de la UPV-EHU de Donostia no lo hay. Gracias a su apoyo, a las becas del Gobierno Vasco (que afortunadamente nunca faltaron) y a trabajar de vez en cuando, pude finalizar los estudios. El segundo cuatrimestre de tercero de carrera cursé Fisiología Animal y recuerdo estar fas- cinado con la asignatura. Tal es así que cuando vi a la profesora Mertxe colgar un anuncio en el que bus- caban candidatos para alumno interno de laboratorio me lancé de cabeza. Y sin fijarme en que era en el departamento de Fisiología, no Fisiología Animal, y en la facultad de Medicina y Odontología, no en la de Ciencias. Seguramente la profesora se limitó a recoger del suelo un anuncio caído. Así que acabé teniendo una entrevista para un grupo del que no tenía ninguna referencia. Y me seleccionaron. A partir de sep- tiembre de 2003 entré como alumno interno bajo la supervisión de María Begoña y José Ignacio, y con ellos obtuve el Diploma de Estudios Avanzados. A ellos les debo mi primer contacto con la ciencia empí- rica, en un departamento muy bien equipado en el que se cuidaba mucho la transmisión del conocimiento y el trabajo en grupo. Allí tuve la posibilidad de trabajar con muy buenos investigadores, algunos amigos hoy en día: Ainara, Ainhoa, Alejandro’, Begoña, Cristian, Fadil, Idoia, Igor, Igotz, Itxaso, Lourdes, Marga, María José, Montse, Nerea, Olatz, Patricia, Rosa, Rosaura, Sandra, Xabier, Yolanda y Yuri (hace tanto que puede que me olvide de alguien). Pero escaseaba la financiación en mi grupo así que empecé a buscar trabajo en neurociencias, campo que siempre me había atraído, y en Madrid, ciudad que me gustaba y conocía de visita. Fue la mujer de Fadil la que, sin conocerme, encontró una oferta en el laboratorio de Maribel y Esther, en el departamento de Farmacología de la facultad de Medicina de la Complutense. Hice una entrevista, me cogieron y me mudé a la capital. Durante esos tres años (enero 2008-diciembre 2010) tuve la suerte de trabajar con Alejandro (postdoc), David, Elizabeth, Erica (postdoc), Icíar, Nieves y Noelia (postdoc). También a Andrés, amigo de la facultad, que se mudó a Madrid cuando le becaron en el mismo departamento. Terminándose ya el contrato, Alejandro me comentó que iba a salir una beca conseguida por el grupo de Psicobiología de la Drogadicción de la UNED (en una convocatoria competitiva del Vicerrectorado de Investigación), grupo en el que él se doctoró. Sólo nos presentamos dos personas y, tras casi siete años de experiencia en laboratorio, yo tenía mejor currículum, así que me seleccionaron. Un año después Ale- jandro se vio codirigiendo mi tesis con Emilio: su primera tesis. Allí también fui agraciado, además de con mis directores, con los compañeros que me encontré, permanentes, duraderos, temporales, estancias, colaboradores, fugaces o amigos: Adrián Emilio, Alberto, Amparo, Ana Belén, Carlos Alberto, Esmeralda, Gonzalo, Gonzalo’, Inmaculada, Inmaculada’, Javier, Jorge Aldrete, Luis, Luis’, Marcos, Mariano, Mario, Miguel, Mónica, Natalia, Nuria, Paula, Raquel, Roberto, Rosa, Santiago, Shishir y Telmo. También pude trabajar, durante una estancia en el instituto Donders (Radboud University, Nijmegen), con Erika, amiga de la carrera que casualmente realizaba la tesis en el grupo de Benno, en el que yo estaba interesado. Por último, Arturo y María Rosa quisieron contratarme, durante mi último año y medio en Madrid (y en España), para trabajar en el departamento de Toxicología de la facultad de Veterinaria de la Complutense, brindándome la posibilidad de conocer a Arantxa, Bernardo, Irma, José Luis, Natalia, Óscar, Óscar’ y Thais. Por casualidades de la vida, pocos meses después de entrar a formar parte del grupo este departamento se fusionó con el de Farmacología de la facultad de Medicina, del que fui miembro años atrás. Llegar hasta aquí no ha sido fácil, y no sólo ha dependido del esfuerzo que yo haya dedicado a mis estu- dios y a mi trabajo: también ha sido gracias a la buena voluntad, a la profesionalidad y a la capacidad de gente que por suerte me he ido encontrando por un camino con altibajos en cuanto a financiación. Por último, agradezco a Dimitris por acompañarme durante todo este difícil proceso, aunque a veces no entendieses muchas cosas del mismo. «Porque los pilares sostienen el templo, pero están separados; y ni el roble ni el ciprés crecen el uno a la sombra del otro». INDEX Abbreviations 1 Abstract 3 1. INTRODUCTION 5 1.1. THE PROBLEM OF DRUGS OF ABUSE AND RELAPSE 7 1.1.1. The problem of relapse 7 1.1.2. Effects of timing on relapse 9 Circadian effects on heroin craving 9 Effect of the frequency of cocaine and alcohol consumption on craving 9 Effect of the abstinence length on craving 10 1.1.3. Clinical relevance of the incubation of craving 11 Research in human craving incubation 11 Clinical relevance in the relapse of drug abuse 12 Clinical relevance in the relapse of food consumption 12 1.2. STATE OF THE ART IN THE INCUBATION OF CRAVING 13 1.2.1. Human neuroimaging studies 13 Studies of fMRI during heroin abstinence 13 Studies of fMRI during food dieting 13 1.2.2. Animal models of craving incubation 14 Animal models of consumption, abstinence and relapse 14 Animal models of seeking incubation 15 1.2.3. Psychobiology of seeking incubation 17 Substances and research groups 17 Biochemical and pharmacological studies in seeking incubation 17 Involvement of brain regions in the incubation of seeking 24 a) Nucleus accumbens 24 b) Amygdala 25 c) Prefrontal cortex 26 d) Dorsal striatum 28 e) Dopaminergic system 29 f) Hippocampus 30 g) Hypothalamus and peptidergic systems 30 h) Serotoninergic system 30 i) Thalamus 31 j) Noradrenergic system 31 Effects of stress and environmental enrichment 31 Effects of sex 33 Effects of age 33 Kinetics of seeking incubation 34 1.2.4. Summary of the state of the art in seeking incubation 34 HYPOTHESIS AND GOALS 37 2. MATERIALS AND METHODS 39 2.1. JUSTIFICATION OF THE MODEL AND THE SAMPLE PROCESING 41 Justification of the model 41 Justification of sample processing 41 2.2. ANIMAL PROCEDURES 42 Animals 42 Surgery 42 Experimental batches 42 Self-administration sessions 42 Missing animals 43 Abstinence and extinction test 43 Behavioural parameters 43 2.3. SAMPLE PROCESING 44 Animal sacrifice 44 Reagents and buffers 44 Brain dissection 44 Missing samples 44 Tissue homogenization 44 2.4. PROTEIN QUANTIFICATION 44 Protein quantification 44 2.5. RADIOIMMUNOASSAY (RIA) 46 Radioimmunoassay 46 2.6. WESTERN BLOT 46 Reagents and buffers 46 Western blot 47 Analysis 47 2.7. CAPILLARY ELECTROPHORESIS 48 Reagents and buffers 48 Capillary electrophoresis 48 Analysis 48 2.8. GENE EXPRESSION 50 RNA isolation 50 RNA quantification 50 Retrotranscription 50 qPCR 50 Analysis 50 2.9. PERINEURONAL NETS 52 Reagents and buffers 52 Fluorescent staining of perineuronal nets 52 Fluorescent microscopy and analysis 52 Issues encountered during the analysis 53 2.10. STATISTIC ANALYSIS 54 Data presentation 54 Univariant statistics 54 Bivariant statistics 54 Principal components analysis (PCA) 54 Structural equation modelling (SEM) 55 3. RESULTS 57 1st Goal: SELF-ADMINISTRATION AND SEEKING INCUBATION 59 3.1. SUBSTANCE SELF-ADMINISTRATION AND SEEKING INCUBATION 59 Incubation of seeking 59 Self-administration behaviour 59 Behavioural predictive variables of seeking incubation 59 2nd Goal: CHANGES IN GLUTAMATERGIC AND GABAERGIC PARAMETERS DURING 61 SEEKING INCUBATION 3.2. SYNAPTIC SCAFOLDING PROTEINS LEVELS 61 Levels of PSD95 and gephyrin by brain region 61 Synaptic coherence between brain regions by SEM 61 Group comparisons 61 3.3. BRAIN AMINES LEVELS 66 Levels of amines in the control groups 66 Changes in amine content 66 3.4. GENE EXPRESSION OF GLUTAMATERGIC, GABAERGIC AND eCB SYSTEMS 68 Gene expression changes in early withdrawal 68 Gene expression changes after protracted withdrawal 69 Gene expression covariation by PCA 71 3.5.
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