Molecular Psychiatry https://doi.org/10.1038/s41380-020-0809-2 ARTICLE Amphetamine sensitization alters hippocampal neuronal morphology and memory and learning behaviors 1,2,5 1,2 1 Luis Enrique Arroyo-García ● Hiram Tendilla-Beltrán ● Rubén Antonio Vázquez-Roque ● 1 3 3 3 1 Erick Ernesto Jurado-Tapia ● Alfonso Díaz ● Patricia Aguilar-Alonso ● Eduardo Brambila ● Eduardo Monjaraz ● 2 4 1 Fidel De La Cruz ● Antonio Rodríguez-Moreno ● Gonzalo Flores Received: 12 November 2019 / Revised: 29 May 2020 / Accepted: 3 June 2020 © The Author(s), under exclusive licence to Springer Nature Limited 2020 Abstract It is known that continuous abuse of amphetamine (AMPH) results in alterations in neuronal structure and cognitive behaviors related to the reward system. However, the impact of AMPH abuse on the hippocampus remains unknown. The aim of this study was to determine the damage caused by AMPH in the hippocampus in an addiction model. We reproduced the AMPH sensitization model proposed by Robinson et al. in 1997 and performed the novel object recognition test (NORt) to evaluate learning and memory behaviors. After the NORt, we performed Golgi–Cox staining, a 1234567890();,: 1234567890();,: stereological cell count, immunohistochemistry to determine the presence of GFAP, CASP3, and MT-III, and evaluated oxidative stress in the hippocampus. We found that AMPH treatment generates impairment in short- and long-term memories and a decrease in neuronal density in the CA1 region of the hippocampus. The morphological test showed an increase in the total dendritic length, but a decrease in the number of mature spines in the CA1 region. GFAP labeling increased in the CA1 region and MT-III increased in the CA1 and CA3 regions. Finally, we found a decrease in Zn concentration in the hippocampus after AMPH treatment. An increase in the dopaminergic tone caused by AMPH sensitization generates oxidative stress, neuronal death, and morphological changes in the hippocampus that affect cognitive behaviors like short- and long-term memories. Introduction the mesolimbic and mesocortical systems [1–3]. Because of its psychostimulant activity, AMPH has been widely used Amphetamine (AMPH) is a central nervous system psy- during the last century in the World Wars, the treatment of chostimulant that triggers monoaminergic release (princi- different diseases, and for hedonic purposes. Nowadays, pally dopamine) from the ventral tegmental area (VTA) to AMPH continues to be used therapeutically in attention deficit hyperactivity disorder and narcolepsy treatments [3]. Its hedonic use has grown considerably, increasing the cost * Gonzalo Flores of health services. For instance, in 2014 around 35.7 million gonzalofl[email protected] people were reported to have used this substance [4], highlighting the interest and importance in investigating the 1 Laboratorio de Neuropsiquiatría, Instituto de Fisiología, effects of AMPH in the brain. Benemérita Universidad Autónoma de Puebla, Puebla, Mexico It has been shown that the repeated AMPH administra- 2 Laboratorio de Fisiología de la Conducta, Escuela Nacional de tion induces changes in regions of high dopaminergic Ciencias Biológicas, Instituto Politécnico Nacional, Ciudad de México, Mexico innervation, causing alterations in neuronal structure, 3 locomotor activity, cognitive behaviors, memory and Facultad de Ciencias Químicas, Benemérita Universidad “ ” Autónoma de Puebla, Puebla, Mexico learning processes, and the pathological craving to con- 4 sume this substance (a theory called incentive sensitization Laboratorio de Neurociencia Celular y Plasticidad, Universidad – Pablo de Olavide, Sevilla, Spain proposed by Robinson and Beridge in 1993, [5 7]). This has been widely studied to understand the underlying neu- 5 Present address: Neuronal Oscillations Laboratory, Division of Neurogeriatrics, Center for Alzheimer Research, NVS, Karolinska ronal mechanisms that generate addiction, withdrawal Institutet, 17164 Solna, Sweden symptoms, or drug relapse [8, 9]. L. E. Arroyo-García et al. Using the incentive sensitization model, it has been are in accordance with the “Guide for Care and Use of found that AMPH affects most of the known brain regions Laboratory Animals” of the Mexican Council for Animal involved in the reward system, due to their role with the Care (Norma Oficial Mexicana NOM-062-ZOO-1999) and development of addiction [5, 10]. However, little is known the European Union Directive 2010/63/EU regarding the about the effect of AMPH on other brain regions like the protection of animals used for scientific purposes, and they hippocampal formation formed by the Cornu Ammonis were approved by the local Ethical Committees. In addition, (CA1, CA2, and CA3), the Dentate Gyrus (DG), the sub- all experiments are in accordance with the ARRIVE iculum, and the entorhinal cortex, [11, 12]. These structures guidelines. All efforts were made to minimize animal suf- contribute to the development of cognitive functions such as fering and to reduce the number of animals used. learning and memory processes and reward-related learning [13, 14]. Interestingly, the VTA projects to the hippo- Amphetamine administration campus, mainly to CA1 [15, 16]. It has also been reported that the repeated administration of dopaminergic psychos- AMPH administration, described previously [5], was used timulants causes impairment in the cognitive functions in order to induce incentive sensitization. Animals were related to hippocampal formation [2, 17, 18]. randomly administered either with D-AMPH or saline Repeated exposure to AMPH probably produces oxida- solution. For AMPH group, the animals were administered tive stress, due to the excessive release of dopamine, which with increasing doses (1–8 mg/kg) of D-AMPH sulfate triggers the production of reactive oxygen species (ROS) (Sigma, St. Louis, Mo, USA) with two intraperitoneal and dopamine quinones [19]. Dysregulation of metabolites injections with the following schema: doses included 1, 2, such as nitric oxide (NO), Zn, malondialdehyde (MDA), 4, 6, and 8 mg/kg, applied for 5 consecutive days, followed and metallothioneins (MTs) has been implicated in cellular by 2 days without administration for 5 weeks. The control damage and morphological changes caused by oxidative group received 1 ml/kg of saline solution. The animals were stress [20–22]. then left undisturbed for 38 days [5] after that time the At present, it is still unclear how AMPH affects cognitive learning and memory test was performed. Once the beha- behaviors and morphological structures in the different vioral test was finished the animals were sacrificed for the brain regions. The spine density changes reported in the experiments (see schematic AMPH administration design, behavioral sensitization protocol are insufficient to explain Fig. 1a). correct neuronal synapses or plasticity. The shapes from these dendritic spines have been correlated with synaptic Learning and memory test strength [23–25], which can be important in neurological disorders [26–28], and has been linked to long-term Onday39afterthelastadministration, tenanimalswere potentiation (LTP) in memory [13]. randomly selected to perform the NORt as previously We determined the long-lasting effect of AMPH sensi- described [28]. It had four phases: habituation, familiar- tization on neuronal morphology using the Golgi–Cox stain, ization, short-term memory test, and long-term memory oxidative stress levels with biochemical procedures, and test, of 6 min for each one, which were recorded using a inflammatory responses using immunohistochemistry and video camera (Sony DCR-SR21, Japan). The experiments neuronal death of the dorsal hippocampus (DH) with ste- were carried out in a Box (60 cm wide × 60 cm high × reological procedures. In addition, possible effects of 60 cm long) for open field. Twenty-four hours after the memory and learning behavior alterations were assessed habituation, animals were exposed to two identical objects using the novel object recognition test (NORt). (familiarization phase) [29]. Then, after 2 h, short-term memory was evaluated changing one of the objects of the previous phase for a novel one. Twenty-four hours after Methods and materials the familiarization phase, in the long-term memory test, the same action was done, changing the former object for Animals a novel one. The time that the animal explored each object was quantified in each phase and a recognition memory Fifty-eight adult male Sprague-Dawley rats (250–350 g) index was calculated in order to assess the time that the were obtained from the Universidad Autónoma de Puebla subject spent recognizing the novel object. The recogni- and Universidad Pablo de Olavide facilities. The animals tion memory index was calculated by the ratio TN/(TK + were kept in a temperature and humidity-controlled envir- TN)inwhichTN is the time spent exploring the novel onment, in a 12–12-h light–dark cycle, with free access to object and TK is the time spent exploring the known water and food. All the procedures described in this study object [29]. Amphetamine sensitization alters hippocampal neuronal morphology and memory and learning behaviors Fig. 1 Experimental design and NORt. Timeline of the amphetamine two equal objects in the familiarization phase, 2 h after the familiar- (AMPH) administration protocol (a). AMPH sensitization impairs ization phase, short-term memory was evaluated with a new object and short- and long-term recognition memory. Short-term memory and 24 h after the last test, long-term memory was