Amphetamine Sensitization Alters Hippocampal Neuronal Morphology and Memory and Learning Behaviors

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 evaluated with a new long-term memory were evaluated using the NORt, which evaluates object. In c) we show the trajectory in each phase for one repre- the exploring time of the new object compared with the familiar one. sentative rat from each group. Error bars are SEM (n = 10 per group). In b) we show the novel recognition index for the ﬁrst contact with *p < 0.05, two-way ANOVA, Bonferroni post-test.

Stereological analysis sections with an area of 2500 μm2 were analyzed [30, 31]. The results were averaged and expressed as the total num- Another group of rats (n = 5 per group) on day 41 after the ber of neurons per region. last administration were anesthetized with sodium pentobarbital (75 mg/kg ip) and perfused with 4% paraf- Golgi–Cox stain method ormaldehyde in 0.1 M phosphate buffer before dissecting the brain. Coronal sections (50 µm thick) from the DH After the behavioral test the animals (n = 10 per group) were stained with cresyl violet were analyzed. A counting grid of deeply anesthetized with sodium pentobarbital (75 mg/kg 250 µm × 250 µm was set inside each contour as a sys- body weight, ip) and perfused intracardially with 0.9% saline tematic random sample procedure with the optical dissector solution. The brains were removed and stained using the fraction (Stereo Investigator System, MicroBrightField modiﬁed Golgi–Coxmethodaspreviouslydescribed[28–32]. Bioscience, USA) using an Olympus BH2 microscope in Coronal sections of 200 µm thickness were obtained from the order to estimate the number of neurons per region (CA1, DH using a vibratome (Campden Instrument, MA752, UK). CA3, and DG). A 50 µm × 50 µm × 50 µm counting frame The sections were maintained in ammonium hydroxide with guard zones of 10 µm was scanned per each XYZ axis for 30 min, followed by 30 min in Kodak Film Fixer, and using a ×60 water immersion objective (UPlanSAPO). ﬁnally rinsed with distilled water and mounted with resinous From each brain at least eight systematically random medium. L. E. Arroyo-García et al.

Microscopic observation and Sholl analysis Metallothioneins (MTs) measurement

The basilar dendritic tree of pyramidal cells from CA1 MT concentration (n = 4 rats per group) was determined (plate 29-37) [33] stained with Golgi–Cox solution were according to the Cd/hemoglobin saturation radioassay [42] drawn. For each animal, neurons from the DH were drawn with some modifications. Briefly, 100 µl of homogenate- using camera lucida at ×400 magnification (DMLS, Leica, supernatant was used. The amount of radioactivity in the Germany) [34]. Dendritic tracings of neural reconstructions supernatant fraction was measured with a Wizard 1470 were quantified by Sholl analysis [35] as reported else- gamma counter (Perkin Elmer, Mexico). MT concentration where, to estimate the total dendritic length and dendritic in tissue is expressed as a µg of MT per g of hippocampus arborization [34, 36–39]. tissue and, was calculated using the molar ratio.

Dendritic spine analyses Malondialdehyde (MDA) analysis

The dendritic spine density was measured as previously MDA concentration (n = 4 rats per group) as a lipid per- reported [35–37, 39–41]. A length of a distal dendrite oxidation marker was determined by formation of thio- (considered distal after the 4th branching order) was barbituric acid reactive substances assay-kit (Cayman traced (at least ≥10 μm long at 1000×), the exact length of Chemical, EUA), following manufacturers instruction [43]. the dendritic segment was calculated, and the number Results were expressed as µM of nitrite per mg of protein. of spines along the dendrite was counted (to yield spines/10 µm). Also, dendritic spines were classified as Immunohistochemistry previously reported [28, 30, 40] into mushroom, thin, stubby, branched, and unclassified spines. In this study For quantitative image analysis of hippocampal immunos- 100 distal dendritic spines per neuron were typified, using taining, sections were processed as reported [29]. Sections five neurons per hemisphere per brain and ten animals were then incubated with GFAP (1:500, Dako, Denmark), per group. CASP3 (1:100, Santa Cruz Biotechnology, USA), MTs-I–II, and MT-III (1:100, Santa Cruz, Biotechnology, USA) as Assay of redox balance primary antibodies, followed by FITC (green color) or Rhodamine (red color) as secondary antibodies (1:100, The hippocampus of six rats per group was dissected, iso- Jackson Immuno Research Laboratories Inc., USA). Photo- lated, and mechanically homogenized in phosphate saline micrographs were taken in CA1, CA3, and DG subfields of buffer, pH 7.4, and centrifuged at 12,500 rpm for 30 min at the hippocampus using a fluorescence microscope (Leica, 4 °C in a TR microcentrifuge (Hanil Science Industrial Co, Germany). The number of immunoreactive cells to GFAP, Ltd; Korea). CASP3, MTs-I–II, and MT-III was quantified using micro- photography corresponding to an area of 500 mm2 [17]. NO assay Statistical analysis NO was determined by evaluating the concentration of nitrites. The nitrites were measured by using the Griess Mean values from each brain region of each animal were reaction [41]. The absorbance of the colorimetric reaction treated as a single measurement for data analysis. All ana- was determined at 540 nm in a spectrophotometer and lyses were carried out by a blind person to experimental compared with a standard curve of NaNO2 (1–10 µM) in conditions in GraphPad Prism 6.0 software. Data for total each assay. Results were expressed as µM of nitrite per mg dendritic length and spine densities were analyzed by “t” of protein. Student, with the AMPH administration considered as an independent factor (p < 0.05 was considered significant). Zn determination Data for the learning and memory test, length per branch order and spine type were analyzed by two-way ANOVA, The hippocampus Zn concentration was assayed using 4- followed by the Bonferroni test for posthoc comparisons, (-2-pyridylazo) resorcinol chromogenic agent [41]. The with time and administration as independent factors for absorbance of the colorimetric reaction was determined learning and memory tests. Branch order and administration using spectrophotometry at 497 nm and compared with a were independent factors for length per branch, and spine standard curve of Zn metal (0.5–5.0 µg/mL water solution) type and administration were independent factors for the in each assay. categorization of spines. Amphetamine sensitization alters hippocampal neuronal morphology and memory and learning behaviors

Results

Amphetamine sensitization impairs short- and long- term memory

AMPH sensitization effects on short- and long-term memory are shown in Fig. 1b, c. In the first phase (familiarization phase), animals of both groups (n = 10 per group) explored both objects for similar periods, which represented a recognition index close to 0.5, no significant difference between these groups (p = 0.9384; Fig. 1b) was observed. In the second phase, the recognition time for the first novel object (short-term memory test) was evaluated 2 h after the familiarization phase. The vehicle group showed a preference for the novel object, whereas the AMPH group showed no preference for any object (p = 0.0484; Fig. 1b). In the third phase (long-term memory test), the subjects were tested 24 h after the second phase. The vehicle-treated group showed a preference for the novel object, unlike the AMPH-treated group, which showed no preference (p = 0.0453; Fig. 1b).

Histological changes on the number of neurons in the hippocampus

Stereological analysis in CA1, CA3, and DG regions showed a reduction in the number of neurons in the CA1 area (Fig. 2a, b). Figure 2a describes histological pictures of the hippocampus in both vehicle and AMPH-treated groups (n = 5 per group). In the CA1 region, a statistical analysis of the average number of cells per region indicates a significant decrease in the number of neurons (t = 3.169, p = Fig. 2 Neuronal density after 35 days of chronic amphetamine (AMPH) administration. Hippocampal sections were stained with 0.0193, Fig. 2b) in the AMPH-treated group compared with cresyl violet and numbers of neurons were counted using the dissector the vehicle-treated group. No changes were observed in the method with digitized images from CA1, CA3, and DG hippocampal CA3 (t = 0.8087, p = 0.4495) or the DG (t = 0.2773, p = regions. In a) representative photomicrographs of the CA1, CA3, and 0.7953) regions (Fig. 2b). These results show that AMPH DG of the dorsal hippocampus from rats treated with vehicle and AMPH are shown, respectively, scale bar, 100 µm. In b) we show the produces a reduction in the number of neurons in CA1, but number of cells for each region, which shows a significant neuron loss not in other hippocampal regions. in the CA1. Error bars are SEM (n = 5 per group). *p < 0.05, unpaired Student’s t test. Amphetamine produces morphological changes in the CA1 hippocampus subfield cells of the AMPH-treated group, compared with their The Golgi–Cox impregnation procedure clearly filled the respective vehicle groups. The dendritic spine density was dendritic shafts of a total of 600 DH pyramidal neurons, the same between groups (p = 0.9580) (Fig. 3a4). The sta- from 10 animals per group. Representative photo- tistical analysis revealed a decrease in mushroom spines micrographs of CA1, CA3, and DG neurons and dendritic (p = 0.0091), thin spines (p = 0.0010), and branched spines spines for the vehicle and AMPH-treated groups are shown (p < 0.0001), and an increase in stubby spines (p < 0.0001) in Fig. 3a1, b1, c1 respectively. The analysis of these data in the CA1 hippocampal region (Fig. 3a5). Regarding the showed an increase in total dendritic length (t = 3.862, p = CA3 subfield, AMPH treatment did not produce changes in 0.0011) in the AMPH-treated group compared with the total dendritic length (Fig. 3b2), dendritic length per branch vehicle group (Fig. 3a2). Furthermore, the arborization test order (Fig. 3b3), and dendritic spine density (Fig. 3b4). showed an increase in the second (p = 0.0004), third (p = Instead, the spine type density test showed a significant 0.0005), and fourth (p = 0.006) branch orders (Fig. 3a3) in decrease in the mushroom type (p < 0.001) between the L. E. Arroyo-García et al.

Fig. 3 Amphetamine (AMPH) sensitization reorganizes the spines; and increases the number of stubby spines (a5). Regarding dendritic arbor as well as the dendritic spine dynamics in the CA3 neurons (b), AMPH does not affect the total dendritic length pyramidal neurons of the hippocampus. Representative photo- (b2), the length per branch order (b3), and the number of spines (b4). micrographs of CA1, CA3, and DG of the dorsal hippocampus neu- However, AMPH reduces the number of mushroom spines (b5). rons (scale bar 50 µm) are shown, as well as dendritic segments (scale Regarding DG neurons (c), AMPH does not affect the total dendritic bar 10 µm) of each group (a1, b1, and c1). Regarding CA1 neurons length (c2), the length per branch order (c3), the number of spines (c4), (a), AMPH increases the total dendritic length (a2), as well as the 2nd, and the dendritic spine populations (c5). Error bars are SEM (n = 10 3rd, and 4th branch orders (a3). AMPH does not affect the number of per group). *p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.001, spines (a4), but reduces the number of mushrooms, thin and bifurcated unpaired Student’s t test and two-way ANOVA, Bonferroni post-test.

AMPH group and the control group, but with no significant groups for MTs (n = 4, p = 0.2379; Fig. 4c) and MDA (n = differences in the other spine types (Fig. 3b5). 4, p = 0.1834; Fig. 4d). Regarding DG granular neurons, AMPH did not have an effect on the total dendritic length of these cells (Fig. 3c2). In GFAP, CASP3, and MTs-III after amphetamine addition, the AMPH treatment did not produce any change in sensitization the arborization (Fig. 3c3). In the same way, neither the analysis of the spine density (Fig. 3c4) nor did the spine type To explain the oxidative stress in the AMPH sensitization, we (Fig. 3c5) reveal any changes between the groups. evaluated the inflammatory response by studying GFAP immunoreactivity, the apoptotic state by means of CASP3 Oxidative stress in amphetamine sensitization immunoreactivity, and the presence of MT-III immunoreactivity, which sequesters heavy metals in the CA1, CA3, To investigate the oxidative stress state after AMPH sensiti- and DG hippocampus subfields (n = 4 per group, Fig. 5a). zation of the hippocampus, the concentrations of NO (n = 6 The number of GFAP immunoreactive cells significantly per group), Zn (n = 6 per group), MDA (n = 4 per group), increased in the CA1 subfield in the AMPH-treated group, and MTs (n = 4 per group) were determined. Forty-one days compared with the vehicle-treated group (p = 0.0149 Fig. 5b). after the last administration of AMPH or saline solution, no In CA3 (p = 0.2269) and DG (p = 0.1363) AMPH subfields differences were found in the nitrite concentration between did not produce a significant difference in the number of groups (p < 0.4395; Fig. 4a). The AMPH-treated group GFAP immunoreactive cells (Fig. 5b). The analysis of the showed a decrease in Zn concentration when compared with number of CASP3 immunoreactive cells did not show a sig- the control group (n = 6, p = 0.0087; Fig. 4b). Conversely, no nificant difference between groups in any region, CA1 (p = differences were found between the AMPH and vehicle 0.2306), CA3 (p = 0.3035), or DG (p = 0.6976) subfields Amphetamine sensitization alters hippocampal neuronal morphology and memory and learning behaviors

Fig. 4 Oxidative response in the hippocampus because of the amphetamine (AMPH) sensitization. The graphs of oxidative biomolecule concentration in micromolar per milligram of protein (µM/mg of protein) are represented in a) for the nitrites (n = 6 per group), in b) for Zn (n = 6 per group), in c) for the metallothioneins (MTs; n = 4 per group). In d) for Malondialdehyde (MDA) concentration was calculated in micromolar per 200 mg of protein (µM/200 mg of protein). Error bars are SEM. **p < 0.01, unpaired Student’s t test.

(Fig. 5c). Regarding MTs-I–II isoforms, AMPH sensitization Even in humans, the exposure to psychostimulants (AMPH did not affect the immunoreactivity of these proteins in any or cocaine) has been linked to deficits in learning and hippocampal subfields (Fig. 5e). Finally, the number of MT- memory processes [46]. Our findings demonstrate that III immunoreactive cells showed a significant increase in the 39 days after the last AMPH administration, performance CA1 (p = 0.0048) and CA3 (p = 0.0315) subfields for the during the NORt is deficient in comparison with the control AMPH-treated group compared with the vehicle-treated group group. Indeed, the AMPH-treated group showed impair- (Fig. 5d). Nevertheless, there was no significant difference ment in both short- and long-term memory tests. NORt between groups in the DG subfield (p = 0.7960, Fig. 5d). evaluates the recognition memory that is necessary to develop the ability to remember [28, 29, 47, 48]. Evidence has shown that malfunction or damage of brain structures Discussion such as the perirhinal cortex and the hippocampal formation are closely related to deficits in recognition memory [47]. Our study indicates that AMPH sensitization induces changes in the brain that persists more than 38 days after the last Amphetamine sensitization reduces the number of administration. Chronic AMPH administration together with cells in the hippocampus the withdrawal syndrome induces deficits in learning and memory behaviors. Furthermore, it produces neuronal death Our study indicates that AMPH sensitization affects the and alterations in neuron morphology in the CA1, CA3, and structures involved in recognition memory, and this damage DG hippocampus subfields. Similarly, inflammatory response lasts for more than a month. For this reason, we evaluated the and oxidative stress in hippocampal formation are apparent. number of cells using the stereological method, since it has These findings show long-lasting damage caused by AMPH been related to cell death and it could indicate the damage in the hippocampus. caused by AMPH in the hippocampus [31, 49]. Our study of the number of cells indicates that chronic exposure to AMPH Amphetamine sensitization impairs memory and induces cell death in the CA1 subfield of the hippocampus, learning processes this result is in agreement with other studies that show that AMPH or methamphetamine causes cell death in this region, Repeated AMPH administration in rodents induces cogni- as well as our previous observations [50]. This result could tive and motor behavior impairments [5, 10, 39, 44, 45]. explain recognition memory impairment and also indicate that L. E. Arroyo-García et al.

the synapses [23–26]. We found no changes in the spine density between groups, but we found shape alterations in the dendritic spines. Thus, mushroom, branched and thin spine types decrease with AMPH treatment, contrary to the stubby type, whose number increases after AMPH treatment. These results support the findings in the NORt, since it has been demonstrated that the mushroom type is the most mature spine with the ability to perform the strongest and most stable synapse [16, 20, 25]. The excitatory synapse begins with a thin immature spine, a thin spine structure is defined as plastic and immature and it transforms into a mushroom type spine in LTP conditions, or the stubby type in LTD protocols [16, 20, 25]. In contrast, the stubby type is described as the spine with a deficient and unstable synapse [24, 26]. Our results suggest that AMPH makes synapses less stable, since the neurons are unable to develop mature spines, which could help to explain the impairment in recognition memory. This effect was specific for the CA1 region, as no changes were found at CA3 or DG (Fig. 3). This further suggests that the hippocampus was affected by AMPH administration and withdrawal. Changes in morphology and cell density that affect learning and memory behaviors can be explained by the generation of oxidative stress [17, 53]. AMPH treatment generates an Fig. 5 Inflammatory response after amphetamine (AMPH) sensi- increase in the dopaminergic tone in the synaptic cleft and tization. Representative photomicrographs of GFAP (green), CASP3 avoids presynaptic dopamine recapture and degradation, (green), MTs-III (red), and MTs-I–II (red) reactivity in CA1, CA3, and GD subfields of the dorsal hippocampus of each group are shown (a). which induces dopamine quinone formation and triggers AMPH only increases the number of GFAP positive cells in CA1 (b). ROS formation, related to damage in the cellular membrane AMPH does not affect the reactivity to CASP3 (c). AMPH increases and apoptosis generation [19]. In our study we evaluated the reactivity to MT-III in CA1 and CA3 subfields (d). AMPH does the presence of nitrites, Zn, MDA, and MTs, 41 days after not affect the reactivity to MTs-I–II (e). Error bars are SEM (n = 4 per group). *p < 0.05, **p < 0.01, unpaired Student’s t test. the last administration to determine the oxidative state in the hippocampus. The results showed that the Zn concentration in the AMPH-treated group decreased compared with the AMPH affects structures with low dopaminergic innervation, vehicle-treated group, but without significant changes in the such as the hippocampus. nitrites, MDAs, and MTs. Nitrites are related to the per- oxynitrite formation, nitrating agents, and strong oxidants Amphetamine sensitization alters the dendritic [22]. The MDA is derived from damaged membrane and dynamic in the hippocampus CA1 lipid peroxidation [22]. These results suggest that in the hippocampus the acute damage caused by the oxidative In previous studies, AMPH sensitization has shown struc- stress is not present 41 days after the last AMPH adminis- tural neuronal alteration in regions with high dopaminergic tration. In addition, this is confirmed by the finding that no innervation, such as the PFC or NAcc. Hypertrophy in the changes in CASP3 immunohistochemistry were observed, dendritic arbor in both of these regions is a form of brain which is a protein responsible for apoptotic processes [54]. sensitization that has been linked to AMPHs [5, 51, 52]. Our findings demonstrate an increase in dendritic length by Amphetamine sensitization causes a Zn deficiency in AMPH treatment in the CA1 region in accordance with the hippocampus what was found by other authors in other regions [5, 51, 52], and that AMPH sensitization induces morpho- Zn is an important mineral with antioxidant properties, logical changes that may be due to neuronal death in this which stabilizes membranes, induces MTs synthesis, med- region. As most of the excitatory synapses are carried out on iates cellular signaling, and acts as a cofactor of enzymes the dendritic spines, establishing the correct neuronal con- and transcriptional genes [55, 56]. Zn deficiency has been nectivity, the dynamics between the number of spines and linked to psychiatric and neurodegenerative disorders spine morphology guarantees the strength and stability of [57, 58]. Our results show a Zn deficiency in the Amphetamine sensitization alters hippocampal neuronal morphology and memory and learning behaviors hippocampus after AMPH-treatment, this result suggests a morphological and biochemical changes in the hippo- dysregulation in the antioxidant molecules that normally campus that persisted for several days after the last protect the hippocampus from oxidative stress. These administration, specifically in CA1. Thus, we suggest that findings highlighted our interest in knowing the MTs con- oxidative stress caused neuronal death and gliosis response centration. Using the biochemical method, we evaluated the in the pyramidal neurons of the CA1 hippocampal region. unspecified MTs in all of the hippocampal formation and Moreover, the increase in the Mts-III levels and the we did not find changes between groups. However, there reduction in Zn levels suggest that the system was unable are four MTs isoforms which are in different tissues of the to modulate the oxidative stress after more than a month of body, the MT-I and MT-II isoforms are in many tissues, the the last AMPH administration. The chronic exposure to MT-III isoform is specifically in the brain and the MT-IV dopaminergic psychostimulants induces ROS and dopa- isoform is expressed in the skin [57, 59]. Our results mine quinone formation [19], corticotrophin-releasing showed that the hippocampus has a low expression of MT-I hormone [66], and redox imbalance [17]. Altogether and MT-II isoforms and no changes were found by AMPH these effects generate oxidative stress that leads to neu- treatment groups (Fig. 5e). Nevertheless, we found an ronal damage in the brain. Furthermore, results from other increase in the expression of MT-III isoform in the hippo- laboratories showed that molecules related to oxidative campus. The increase in MT concentrations has been related stress are affected after 1 month of forced withdrawal in to chronic inflammation. Evidence suggests that pro- regions of high dopaminergic innervation [66]. We suggest inflammatory cytokines induce the increase in MT con- that the neuronal death found in the hippocampal CA1 centration, which generate a subsequental increase in Zn region may be caused by its vulnerability to oxidative sequestration [60]. This may explain our findings in the Zn stress [64, 65]. The morphological changes in the CA1 and MT concentration and also suggest a chronic inflam- region suggest a plastic compensation to the neuronal mation in the hippocampus. death that is not completely functional. The deficiency of Zn and the increase in the reactive astrocytes found in our Amphetamine sensitization increases GFAP model, plus the hypothetic imbalance in corticotrophin- immunoreactivity in the hippocampus releasing hormone and trophic factors [66] could interfere with the correct maturation of the dendritic spine mor- We found an increase in the number of reactive astrocytes phology. All of these changes may be related to short and in the AMPH-treated subjects in the CA1 hippocampus long-term memory deficits also observed in the AMPH subfield. Previous results from our laboratory demon- sensitization animal model. Consequently, our results strated that the dopamine agonist induced the increase in suggest that the AMPH sensitization model generates reactive astrocytes in the hippocampus [17]. The reactive oxidative stress, neuronal death, gliosis, and reduced gliosis is expressed during brain damage and oxidative synaptic communication in the hippocampus that affects stress to maintain cellular integrity, reducing the spread memory processes. of inflammation and damage. But also, these reactive astrocytes have demonstrated that preventing axonal Acknowledgements LEA-G and HT-B acknowledge CONACYT for regeneration can affect the functionality of the brain [61]. the fellowship. EB, PA-A, AD, RAV-R, FDLC, EM, and GF acknowledge the “Sistema Nacional de Investigadores” of Mexico for Our results from reactive astrocytes match the number of memberships. Thanks to Miguel Tapia-Rodríguez (Instituto de cells found in the CA1, which explains the inflammation Investigaciones Biomédicas, Universidad Nacional Autónoma de and damage observed in this area and may well explain México) for stereological procedures assistance and to Professor the malfunction in recognition memory. In fact, several Robert Simpson for editing the English language text. reports suggest that CA1 is a hippocampal region with a Funding – Funding for this study was provided by grants from the high density expression of D1 D5 receptors, which could PRODEP (CA-BUAP-120) and the CONACYT grant (No. 252808) to explain the loss of cells, an increase in GFAP expression GF and MINECO/FEDER (BFU2012-38208) and the Junta de in this region and most of the damage found in the hip- Andalucía (P11-CVI-7290) to AR-M. None of the funding institutions pocampus [14, 62, 63]. Moreover, in vitro [64]and had any further role in the study design, the collection or interpretation of data, analyses, the writing of the report or the decision to submit the in vivo [65] studies have demonstrated the CA1 vulner- paper for publication. ability to oxidative stress, which explains the selective AMPHeffectstothisarea. Author contributions LEA-G, HT-B, AR-M, RAV-R, FDLC, and GF In summary, we hypothesized that chronic AMPH designed the study and wrote the protocol. LEA-G, HT-B, EEJT, AD, administration and the subsequent withdrawal syndrome PA-A, EB, and EM performed the experiments. LEA-G, AR-M, and GF performed the literature searches and analysis and LEA-G and GF produce a sustained oxidative state affecting the brain undertook the statistical analysis. LEA-G, AR-M, and GF wrote the structures with dopaminergic innervation. Our results first draft of the manuscript. All contributing authors have approved showed that the AMPH sensitization model induced the final manuscript. L. E. Arroyo-García et al.

Compliance with ethical standards 20. Eibl JK, Abdallah Z, Ross GM. Zinc-metallothionein: a potential mediator of antioxidant defence mechanisms in response to – Conflict of interest The authors declare that they have no conflict of dopamine induced stress. Can J Physiol Pharm. 2010;88:305 12. interest. 21. Cuajungco MP, Lees GJ. Zinc metabolism in the brain: relevance to human neurodegenerative disorders. Neurobiol Dis. 1997;4:137–69. 22. Wigner P, Czarny P, Galecki P, Su KP, Sliwinski T. The mole- Publisher’s note Springer Nature remains neutral with regard to cular aspects of oxidative & nitrosative stress and the tryptophan jurisdictional claims in published maps and institutional affiliations. catabolites pathway (TRYCATs) as potential causes of depression. Psychiatry Res. 2018;262:566–74. 23. Hayashi Y, Majewska AK. Dendritic spine geometry: functional References implication and regulation. Neuron. 2005;46:529–32. 24. Segal M. Dendritic spines, synaptic plasticity and neuronal sur- 1. Cao DN, Shi JJ, Hao W, Wu N, Li J. Advances and challenges in vival: activity shapes dendritic spines to enhance neuronal viabi- pharmacotherapeutics for amphetamine-type stimulants addiction. lity. Eur J Neurosci. 2010;31:2178–84. Eur J Pharm. 2016;780:129–35. 25. Pérez-Rodríguez M, Arroyo-García LE, Prius-Mengual J, Andrade- 2. Cadet JL, Bisagno V, Milroy CM. Neuropathology of substance Talavera Y, Armengol JA, Pérez-Villegas E, et al. Adenosine use disorders. Acta Neuropathol. 2014;127:91–107. receptor-mediated developmental loss of spike timing-dependent 3. Sulzer D, Sonders MS, Poulsen NW, Galli A. Mechanisms depression in the hippocampus. Cereb Cortex. 2019;29:3266–3281. of neurotransmitter release by amphetamines: a review. Prog Neu- 26. Fiala JC, Spacek J, Harris KM. Dendritic spine pathology: cause robiol. 2005;75:406–33. or consequence of neurological disorders? Brain Res Brain Res 4. United Nations. World Drug Report 2016. New York: United Rev. 2002;39:29–54. Nations press; 2016. 27. Blanpied TA, Ehlers MD. Microanatomy of dendritic spines: 5. Robinson TE, Kolb B. Persistent structural modifications in emerging principles of synaptic pathology in psychiatric and neu- nucleus accumbens and prefrontal cortex neurons produced by rological disease. Biol Psychiatry. 2004;55:1121–7. previous experience with amphetamine. J Neurosci. 1997;17: 28. Tendilla-Beltrán H, Antonio Vázquez-Roque R, Judith Vázquez- 8491–7. Hernández A, Garcés-Ramírez L, Flores G. Exploring the den- 6. Robinson TE, Berridge KC. The neural basis of drug craving: an dritic spine pathology in a schizophrenia-related neurodevelop- incentive-sensitization theory of addiction. Brain Res Brain Res mental animal model. Neuroscience. 2019;396:36–45. Rev. 1993;18:247–91. 29. Hernandez-Hernandez EM, Caporal Hernández K, Vázquez- 7. Vanderschuren LJ, Pierce RC. Sensitization processes in drug Roque RA, Díaz A, de la Cruz F, Flores G. The neuropeptide- addiction. Curr Top Behav Neurosci. 2010;3:179–95. 12 improves recognition memory and neural plasticity of the 8. Robinson TE, Berridge KC. Review. The incentive sensitization limbic system in old rats. Synapse. 2018;72:e22036. theory of addiction: some current issues. Philos Trans R Soc Lond 30. Tendilla-Beltrán H, Meneses-Prado S, Vázquez-Roque RA, Tapia- B Biol Sci. 2008;363:3137–46. Rodríguez M, Vázquez-Hernández AJ, Coatl-Cuaya H, et al. Ris- 9. Robinson TE, Berridge KC. Addiction. Annu Rev Psychol. 2003; peridone ameliorates prefrontal cortex neural atrophy and oxidative/ 54:25–53. nitrosative stress in brain and peripheral blood of rats with neonatal 10. Hedou G, Homberg J, Feldon J, Heidbreder CA. Expression of ventral hippocampus lesion. J Neurosci. 2019;39:8584–99. sensitization to amphetamine and dynamics of dopamine neuro- 31. Vázquez‐Roque RA, Ramos B, Tecuatl C, Juárez I, Adame A, de transmission in different laminae of the rat medial prefrontal la Cruz, et al. Chronic administration of the neurotrophic agent cortex. Neuropharmacology. 2001;40:366–82. cerebrolysin ameliorates the behavioral and morphological chan- 11. Andersen P, Morris R, Amaral DG, Bliss T, O´Keefe J. The ges induced by neonatal ventral hippocampus lesion in a rat model hippocampus book. New York: Oxford University Press; 2007. of schizophrenia. J Neurosci Res. 2012;90:288–306. 12. Volkow ND, Wang GJ, Fowler JS, Tomasi D. Addiction circuitry 32. Gibb R, Kolb B. A method for vibratome sectioning of Golgi-Cox in the human brain. Annu Rev Pharm Toxicol. 2012;52:321–36. stained whole rat brain. J Neurosci Methods. 1998;79:1–4. 13. Broadbent NJ, Gaskin S, Squire LR, Clark RE. Object recognition 33. Paxinos G, Watson C. The rat brain in stereotaxic coordinates, 2nd memory and the rodent hippocampus. Learn Mem. 2010;17:5–11. edn. New York: Academic Press; 1986. 14. Rosen ZB, Cheung S, Siegelbaum SA. Midbrain dopamine neu- 34. Kolb B, Forgie M, Gibb R, Gorny G, Rowntree S. Age, experience rons bidirectionally regulate CA3-CA1 synaptic drive. Nat Neu- and the changing brain. Neurosci Biobehav Rev. 1998;22:143–59. rosci. 2015;18:1763–71. 35. Sholl DA. Dendritic organization in the neurons of the visual and 15. McNamara CG, Dupret D. Two sources of dopamine for the motor cortices of the cat. J Anat. 1953;87:387–406. hippocampus. Trends Neurosc. 2017;40:383–4. 36. Flores G, Alquicer G, Silva-Gómez AB, Zaldivar G, Stewart J, 16. Scofield MD, Heinsbroek JA, Gipson CD, Kupchik YM, Spencer Quirion R, et al. Alterations in dendritic morphology of prefrontal S, Smith ACW, et al. The nucleus accumbens: mechanisms of cortical and nucleus accumbens neurons in post-pubertal rats after addiction across drug classes reflect the importance of glutamate neonatal excitotoxic lesions of the ventral hippocampus. Neu- homeostasis. Pharm Rev. 2016;68:816–71. roscience. 2005;133:463–70. 17. Arroyo-García LE, Vázquez-Roque RA, Díaz A, Treviño S, De 37. Martínez-Tellez R, Gómez-Villalobos MJ, Flores G. Alteration in La Cruz F, Flores G, et al. The effects of non-selective dopamine dendritic morphology of cortical neurons in rats with diabetes receptor activation by apomorphine in the mouse hippocampus. mellitus induced by streptozocin. Brain Res. 2005;1048:108–15. Mol Neurobiol. 2018;55:8625–36. 38. Silva-Gómez AB, Bermudez M, Quirion R, Srivastava LK, Picazo 18. Arroyo-Garcia LE, Rodríguez-Moreno A, Flores G. Apomorphine O, Flores G. Comparative behavioral changes between male and effects on the hippocampus. Neural Regeneration Res. 2018;13: female postpubertal rats following neonatal excitotoxic lesions of 2064–6. the ventral hippocampus. Brain Res. 2003;973:285–92. 19. Cadet JL, Krasnova IN, Jayanthi S, Lyles J. Neurotoxicity of 39. Tendilla-Beltrán H, Arroyo-García LE, Díaz A, Camacho-Abrego I, substituted amphetamines: molecular and cellular mechanisms. de la Cruz F, Rodriguez-Moreno A, et al. The effects of ampheta- Neurotox Res. 2007;11:183–202. mine exposure on juvenile rats on the neuronal morphology of the Amphetamine sensitization alters hippocampal neuronal morphology and memory and learning behaviors

limbic system at prepubertal, pubertal and postpubertal ages. J Chem than nonassociative drug sensitization. Biol Psychiatry. 2009;65: Neuroanat. 2016;77:68–77. 835–40. 40. Bello-Medina PC, Flores G, Quirarte GL, McGaugh JL, Prado- 53. Regev L, Baram TZ. Corticotropin releasing factor in neuroplas- Alcalá RA. Mushroom spine dynamics in medium spiny neurons ticity. Front Neuroendocrinol. 2014;35:171–9. of dorsal striatum associated with memory of moderate and 54. Mirzayans R, Andrais B, Kumar P, Murray D. The growing com- intense training. Proc Natl Acad Sci USA 2016;113:E6516–25. plexity of cancer cell response to DNA-damaging agents: caspase 3 41. Tellez-Merlo G, Morales-Medina JC, Camacho-Abrego I, Juaréz- mediates cell death or survival? Int J Mol Sci. 2016;17:708. Díaz I, Aguilar-Alonso P, de la Cruz F, et al. Prenatal immune 55. Marreiro DD, Cruz KJ, Morais JB, Beserra JB, Severo JS, de challenge induces behavioral deficits, neuronal remodeling, and Oliveira AR. Zinc and oxidative stress: current mechanisms. increases brain nitric oxide and zinc levels in the male rat off- Antioxidants. 2017;6. spring. Neuroscience. 2019;406:594–605. 56. Travaglia A, La Mendola D, Magrì A, Pietropaolo A, Nicoletti VG, 42. Eaton DL, Toal BF. Evaluation of the Cd/hemoglobin affinity Grasso G, et al. Zinc(II) interactions with brain-derived neurotrophic assay for the rapid determination of metallothionein in biological factor N-terminal peptide fragments: inorganic features and biolo- tissues. Toxicol Appl Pharm. 1982;66:134–42. gical perspectives. Inorg Chem. 2013;52:11075–83. 43. Ohkawa H, Ohishi N, Yagi K. Assay for lipid peroxides in animal 57. Szewczyk B. Zinc homeostasis and neurodegenerative disorders. tissues by thiobarbituric acid reaction. Anal Biochem. 1979;95:351–8. Front Aging Neurosci. 2013;5:33. 44. Tindell AJ, Berridge KC, Jun Z, Peciña S, Aldridge JW. Ventral 58. Baltaci AK, Yuce K, Mogulkoc R. Zinc metabolism and metal- pallidal neurons code incentive motivation: amplification by lothioneins. Biol Tra Ele Rese. 2017;55:223–33. mesolimbic sensitization and amphetamine. Eur J Neurosci. 2005; 59. Juárez-Rebollar D, Rios C, Nava-Ruíz C, Méndez-Armenta M. 22:2617–34. Metallothionein in brain disorders. Oxid Med Cell Longev. 45. Zhu J, Chen Y, Zhao N, Cao G, Dang Y, Chen T. Distinct roles of 2017;2017:5828056. dopamine D3 receptors in modulating methamphetamine-induced 60. Vasto S, Mocchegiani E, Malavolta M, Cuppari I, Listi F, Nuzzo behavioral sensitization and ultrastructural plasticity in the shell of D, et al. Zinc and inflammatory/immune response in aging. Ann N the nucleus accumbens. J Neurosci Res. 2012;90:895–904. Y Acad Sci. 2007;1100:111–22. 46. Reske M, Eidt CA, Delis DC, Paulus MP. Nondependent stimu- 61. Kang K, Lee SW, Han JE, Choi JW, Song MR. The complex lant users of cocaine and prescription amphetamines show verbal morphology of reactive astrocytes controlled by fibroblast growth learning and memory deficits. Biol Psychiatry. 2010;68:762–9. factor signaling. Glia. 2014;62:1328. 47. Brown MW, Aggleton JP. Recognition memory: what are 62. Yang K, Broussard JI, Levine AT, Jenson D, Arenkiel BR, Dani the roles of the perirhinal cortex and hippocampus? Nat Rev JA. Dopamine receptor activity participates in hippocampal Neurosci. 2001;2:51–61. synaptic plasticity associated with novel object recognition. Eur J 48. Bevins RA, Besheer J. Object recognition in rats and mice: a one- Neurosci. 2017;45:138–46. trial non-matching-to-sample learning task to study ‘recognition 63. Sjulson L, Peyrache A, Cumpelik A, Cassataro D, Buzsáki G. memory’. Nat Protoc. 2006;1:1306–11. Cocaine place conditioning strengthens location-specifichip- 49. Kristiansen SL, Nyengaard JR. Digital stereology in neuro- pocampal coupling to the nucleus accumbens. Neuron. 2018;98: pathology. APMIS. 2012;120:327–40. 926–34.e5. 50. Kuczenski R, Everall IP, Crews L, Adame A, Grant I, Masliah E. 64. Wang X, Pal R, Chen XW, Limpeanchob N, Kumar KN, Escalating dose-multiple binge methamphetamine exposure Michaelis EK. High intrinsic oxidative stress may underlie results in degeneration of the neocortex and limbic system in the selective vulnerability of the hippocampal CA1 region. Brain rat. Exp Neurol 2007;207:42–51. Res. 2005;140:120–6. 51. Crombag HS, Gorny G, Li Y, Kolb B, Robinson TE. Opposite 65. Wilde GJ, Pringle AK, Wright P, Iannotti F. Differential vulnerability effects of amphetamine self-administration experience on den- of the CA1 and CA3 subfields of the hippocampus to superoxide and dritic spines in the medial and orbital prefrontal cortex. Cereb hydroxyl radicals in vitro. J Neurochem. 1997;69:883–6. Cortex. 2005;15:341–8. 66. Cadet JL, Patel R, Jayanthi S. Compulsive methamphetamine 52. Singer BF, Tanabe LM, Gorny G, Jake-Matthews C, Li Y, Kolb taking and abstinence in the presence of adverse consequences: B, et al. Amphetamine-induced changes in dendritic morphology epigenetic and transcriptional consequences in the rat brain. in rat forebrain correspond to associative drug conditioning rather Pharm Biochem Behav. 2019;179:98–108.