The Acute Activation of the CB1 Receptor in the Hippocampus Decreases Neurotoxicity and Prevents Spatial Memory Impairment in Rats Lesioned with β-Amyloid 25–35

NEUROSCIENCE RESEARCH ARTICLE A. Patricio-Martı´nez et al. / Neuroscience 416 (2019) 239–254

The Acute Activation of the CB1 Receptor in the Hippocampus Decreases Neurotoxicity and Prevents Spatial Memory Impairment in Rats Lesioned with b-Amyloid 25–35 Aleidy Patricio-Martı´ nez, a,b Rodolfo Sa´nchez-Zavaleta, a Isael Angulo-Cruz, a Liliana Gutierrez-Praxedis, a Eleazar Ramı´ rez, a Isabel Martı´ nez-Garcı´ a c and Ilhuicamina Daniel Limo´n a,* a

Laboratorio de Neurofarmacologı´a, Facultad de Ciencias Quı´micas-Beneme´rita Universidad Auto´noma de Puebla, Puebla, Mexico

b

Facultad de Ciencias Biolo´gicas-Beneme´rita Universidad Auto´noma de Puebla, Puebla, Mexico

c

Laboratorio de Neuroquı´mica, Facultad de Ciencias Quı´micas-Beneme´rita Universidad Auto´noma de Puebla, Puebla, Mexico

Abstract—Given their anti-inflammatory properties, cannabinoids have been shown to be neuroprotective agents and to reduce excitotoxicity, through the activation of the Cannabinoid receptor type 1 (CB1r). These properties have led to CB1r being proposed as pharmacological targets for the treatment of various neurodegenerative diseases. Amyloid-b 25–35 (Ab25–35) induces the expression of inducible nitric oxide synthase (iNOS) and increases nitric oxide (NO) levels. It has been observed that increased NO concentrations trigger biochemical pathways that contribute to neuronal death and cognitive damage. This study aimed to evaluate the neuroprotective effect of an acute activation of CB1r on spatial memory and its impact on iNOS protein expression, NO levels, gliosis and the neurodegenerative process induced by the injection of Ab(25–35) into the CA1 subfield of the hippocampus. ACEA [1 lM/1 lL] and Ab(25–35) [100 lM/1 lL] and their respective vehicle groups were injected into the CA1 subfield of the hippocampus. The animals were tested for spatial learning and memory in the eight-arm radial maze, with the results revealing that the administration of ACEA plus Ab(25–35) improves learning and memory processes, in contrast with the Ab(25–35) group. Moreover, ACEA plus Ab(25–35) prevented both the increase in iNOS protein and NO levels and the reactive gliosis induced by Ab(25–35). Importantly, neurodegeneration was significantly reduced by the administration of ACEA plus Ab(25–35) in the CA1 subfield of the hippocampus. The data obtained in the present research suggest that the acute early activation of CB1r is crucial for neuroprotection. Ó 2019 IBRO. Published by Elsevier Ltd. All rights reserved.

Key words: amyloid-b(25–35); hippocampus; CB1 receptor; iNOS; neurodegeneration; spatial memory.

Ab are 40 and 42 residues in length (Ab1–40 and Ab1–42, respectively) and are produced by the processing of amyloid precursor protein (APP) (Younkin, 1998). Ab oligomers can trigger cascade neurotoxicity and neurodegeneration (Lambert et al., 1998; Walsh et al., 2002).The toxic properties of the native full-length Ab(1–42) peptide are retained in the 25–35 fraction of amyloid-b (Ab25–35) (Pike et al., 1995; Butterfield and Boyd-Kimball, 2005). However, the neurotoxic effects generated by the Ab(25– 35) peptide are more rapidly and cause more oxidative damage compared to those generated by Ab(1–42) (Varadarajan et al., 2001). The amyloid hypothesis of AD posits that, regardless of whether the disease is familial or sporadic, the accumulation and aggregation of different amyloid peptides in the brain lead to the formation of senile plaques (Selkoe, 2000; Hardy and Selkoe, 2002). The experimental intracerebral or intracerebroventricular infusion of amyloid peptides can mimic some aspects of AD (Maurice et al., 1996; Meunier et al., 2006; Lawlor and

INTRODUCTION Alzheimer’s disease (AD) is the most common neurodegenerative disease in the world, generally affecting people aged 65 and older (WHO, 2012). This pathology is characterized by progressive memory loss and the formation of extracellular amyloid-b peptide (Ab) deposits, leading to the formation of neuritic plaques and neurofibrillary tangles in the hippocampus and the cortex (Maccioni et al., 2001). The two major forms of *Corresponding author at: Laboratorio de Neurofarmacologı´ a, FCQBeneme´rita Universidad Auto´noma de Puebla, 14 Sur y Av. San Claudio C.U. Edificio FCQ-3, A.P. 72570, Puebla, Puebla, Mexico. Tel.: +52 222 229 55 00x7528. E-mail address: [email protected] (I. D. Limo´n). Abbreviations: ACEA, arachidonyl-2-chloroethylamide; AD, Alzheimer’s disease; ANOVA, one-way analysis of variance; APP, amyloid precursor protein; Ab(35–25), amyloid-b 35–25; Ab(25–35), amyloid-b 25–35; CB1r, Cannabinoid receptor 1; CB2r, Cannabinoid receptor 2; GFAP, Glial fibrillary acidic protein; Iba-1, Ionized calciumbinding adaptor molecule 1; iNOS, inducible nitric oxide synthase; NO, nitric oxide. https://doi.org/10.1016/j.neuroscience.2019.08.001 0306-4522/Ó 2019 IBRO. Published by Elsevier Ltd. All rights reserved. 239

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Young, 2010; Zussy et al., 2011). The Ab peptide fragments (Ab1–40, Ab1–42 or Ab25–35) can be administered acutely, either by stereotactic injection (Harkany et al., 1998, 2000) or continuous infusion (Harkany et al., 2000; Olariu et al., 2002; Yamada et al., 2005). It has been shown that the direct intracerebral injection of amyloid peptides into different brain regions causes a deficit in learning and memory processes, as well as behavioral alterations similar to those observed in AD (Maurice et al., 1996; Harkany et al., 1998; Stepanichev et al., 2003; Yamada et al., 2005; Sipos et al., 2007; Limo´n et al., 2009a,b; Dı´ az et al., 2012; Ramı´ rez et al., 2019). Although the complete complexity of the pathology is not reproduced in humans, the intracerebral administration of Ab peptides has been associated with inflammatory response, oxidative stress, apoptosis, synaptic reduction and the moderate loss of cholinergic and glutamatergic neuronal cells (Harkany et al., 1998, 2000; Weldon et al., 1998; Ortega et al., 2014; PatricioMartı´ nez et al., 2016; Ramı´ rez et al., 2018). Some advantages of the use of Ab infusion models in rodents are that they enable the administration of defined amounts of a specific Ab and, furthermore, yield experimental results in a few weeks (one to two) instead of requiring several months for the development of an aged transgenic model (Frautschy et al., 1996; Maurice et al., 1996; Klementiev et al., 2007; Chavant et al., 2010; Dı´ az et al., 2010). In particular, Ab(25–35) could be responsible for toxic and oxidative events, such as oxidative stress-mediated changes, that lead to brain damage (Gulyaeva and Stepanichev, 2010). Previous work by our research group has shown that the administration of the Ab(25–35) peptide in the CA1 subfield of the hippocampus, temporal cortex and medial septum of rats produces a considerable decline in learning and spatial memory (Limo´n et al., 2009a,b; Dı´ az et al., 2012; Ortega et al., 2014; PatricioMartı´ nez et al., 2016). These events are associated with the activation of the NMDA receptor, which facilitates a massive input of calcium, thus inducing cholinergic toxicity, which, in turn, involves oxidative and nitrosative stress, apoptosis, and neuroinflammation, resulting in neuronal death (Harkany et al., 1999, 2000; Parks et al., 2001; Dı´ az et al., 2012; Ortega et al., 2014; Stepanichev et al., 2014). The pro-inflammatory response to Ab(25–35), mediated by gliosis (astrocytes and microglia), induces the expression of inducible nitric oxide synthase (iNOS), an enzyme responsible for increasing nitric oxide (NO) to neurotoxic levels. iNOS is crucial in the formation of NO, as the specific inhibition of iNOS decreases NO concentrations (Dı´ az et al., 2011, 2014). It has been observed that an increase in NO concentrations triggers biochemical pathways that contribute to neuronal death and cognitive damage (Limo´n et al., 2009a; Dı´ az et al., 2010). The cannabinoid system has been proposed as a promising therapeutic goal in the treatment of AD, as it has the ability to modulate several mechanisms present in both the pathology and different neurodegeneration models (Devinsky et al., 2014; Han et al., 2014). Type 1 cannabinoid receptor (CB1r) is Gi/o protein-coupled and

widely expressed in the brain (Herkenham et al., 1991), predominantly in the neurons of the cerebellum and the basal ganglia, but also in glial cells and peripheral tissue (Piomelli, 2003; Stella, 2009, 2010; Hu and Mackie, 2015). Type 2 cannabinoid receptors (CB2rs) are also Gi/o protein-coupled and display a distinct expression pattern in the cells and tissues of the immune system; however, it has recently been shown that CB2r is expressed in the central nervous system (CNS), specifically in astrocytes, microglia and neurons (Begg et al., 2005; Stella, 2010; Atwood and Mackie, 2010; Lou et al., 2011). In situ hybridization studies show that CB2r mRNAs are expressed in the neurons of the cerebellum (Skaper et al., 1996), internal globus pallidus (Zhang et al., 2015), ventral tegmental area (Zhang et al., 2014), nucleus accumbens, dorsal striatum (Zhang et al., 2015), cerebral cortex and hippocampus (Lanciego et al., 2011; Sierra et al., 2015). While the expression of CB2r in the brain is the subject of much debate, it has been proposed that these receptors are absent in healthy brains (Munro et al., 1993; Derocq et al., 1995; Griffin et al., 1999; Sugiura et al., 2000; Carlisle et al., 2002; Stella, 2004; Manzanares et al., 2018), while, CB2r can be detected in the microglia, associated with neuritic plaque, in the brain tissue of patients with AD (Benito et al., 2003). Several studies have demonstrated that natural and synthetic exogenous cannabinoids exert a neuroprotective action, via CB1/CB2r, both against the neurotoxicity process and in the induction of repair mechanisms in response to neuronal damage (El-Remessy et al., 2003; Marsicano et al., 2003; Harvey et al., 2012). As CB1r activation results in the inhibition of the activation of adenylate cyclase and calcium flux at the axonal terminal, CB1r signaling suppresses the release of neurotransmitters at the synapse (Freund et al., 2003). Both the synthetic cannabinoid WIN-55212-2 (non-elective CB1/CB2r agonist) and D-9tetrahydrocannabinol (D9-THC) have been shown to exert neuroprotective effects on glutamate toxicity in rat hippocampal neurons; moreover, they also inhibit Nand P/Q-type calcium (Ca2+) channels and decrease oxidative stress (Shen and Thayer, 1998; Marsicano et al., 2002; Gilbert et al., 2007). In addition, WIN55212-2 is able to generate anti-inflammatory and cognitive-enhancing effects, and induce neurogenesis during both normal and pathological aging in rats (Marchalant et al., 2008, 2009). Similarly, D9-THC decreases the toxicity induced by kainic acid in spinal cord cultures, thus improving cell viability (Abood et al., 2001). These neuroprotective properties have been linked to the ability of CB1r to suppress glutamatergic activity (Shen et al., 1996; Takahashi and Castillo, 2006). At a behavioral level, it has been reported that the chronic activation of CB1r by ACEA agonist reduces the cognitive impairment observed in double transgenic mice (AbPP/PS1) from 6 months of age onwards (Aso et al., 2012). The 7-day intracerebroventricular administration of the synthetic cannabinoid WIN55-212-2 in rats

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prevents both the activation of the microglia and the cognitive deterioration induced by the Ab(25–35) peptide (Ramirez et al., 2005). CB2r is highly inducible, mainly in the microglia, under certain conditions, such as after brain injuries (Atwood and Mackie, 2010). CB2r modulates microglial migration and infiltration in areas of the brain presenting neuroinflammation and degeneration (Walter et al., 2003; Nun˜ez et al., 2004). It has been proposed that, in addition to regulating inflammatory processes, CB2r plays an important role in neural proliferation, axon guidance (Palazuelos et al., 2012; Duff et al., 2013) and synaptic transmission (Kim and Li, 2015; Li and Kim, 2016). In vitro experiments have shown that selective CB2r agonists, such as JWH-015, JWH-133 and HU-308, in contrast to mixed CB1/CB2r agonists, such as WIN-55212-2 and HU-210, both decrease the release of pro-inflammatory cytokines and restrict the neurodegenerative process in cultures of exposed microglial cells with different fractions of the Ab peptide (Ehrhart et al., 2005; Ramirez et al., 2005; Martı´ n-Moreno et al., 2011). Using a selective CB2r agonist, Esposito et al. (2007) show that the astrocytic proliferation induced by Ab is facilitated in cell cultures, findings corroborated in vivo via the administration of the selective agonists CB2r and CB1/CB2r in rats and mice administered intracerebrally with Ab. Both the levels of proinflammatory cytosines and the reactivity of the microglia were shown to decrease (Ramirez et al., 2005; Esposito et al., 2007; Martı´ n-Moreno et al., 2011; Fakhfouri et al., 2012; Wu et al., 2013). Cannabinoids have been shown to induce antiinflammatory activity that may involve NO signaling. In addition, the treatment of cell cultures with cannabidiol prior to the deposition of Ab(1–42) significantly increases cell survival and decreases oxidative stress, caspase 3 levels, and intracellular concentrations of the Ca2+ ion (Iuvone et al., 2004). Esposito et al. (2006) observed that the administration of ACEA (a selective CB1r agonist) is able to inhibit the expression of iNOS and NO levels in C6 line cultures. The evidence shows that chronic administrations of CB1r agonists have an important role, not only in the regulation of the inflammatory process but also in promoting intracellular pathways that promote restoration, apoptotic inhibition and, consequently, neurodegeneration. The present study evaluated the neuroprotective effect of the acute administration of ACEA on iNOS protein expression, NO levels, gliosis and the neurodegenerative process induced by the injection of Ab(25–35) into the CA1 subfield of the hippocampus. It also sought to ascertain whether this treatment has an effect on spatial learning and memory processes in rats.

EXPERIMENTAL PROCEDURES Subjects Adult male Wistar rats (260–300 g) (n = 96) were obtained from the Claude Bernard animal facilities at the Beneme´rita Universidad Auto´noma de Puebla (BUAP). They were individually housed in groups of four–six per

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cage in constant temperature conditions of 22 ± 2 °C and a 12-h light/dark cycle (lights on at 8 am), with food and water ad libitum. All experimental procedures conformed with the Guide for Care and Use of Laboratory Animals set out in NOM-062-ZOO-1999 (Norma Oficial Mexicana, or Official Mexican Standard) and were approved by the BUAP bioethics committee. Drug treatments The selective CB1r agonist arachidonyl-2chloroethylamide (ACEA) (Tocris Cookson Inc. Ballwin, MO) and the CB1r antagonist N-(Piperidin-1-yl)-5-(4-iodo phenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-c arboxamide (AM-251) (Sigma-Aldrich Ltd., St. Louis, MO, USA) were dissolved in 0.1% dimethyl sulfoxide (DMSO) to a final concentration of 1 lM and 10 lM, respectively (Mun˜oz-Arenas et al., 2015). All drug solutions were freshly prepared before use, while each group received a bilateral intracerebral microinjection into the CA1 subfield of the hippocampus. Ab(25–35) aggregation The Ab(35–25) and Ab(25–35) fractions (Sigma-Aldrich Ltd., St. Louis, MO, USA) were dissolved in phosphate buffered saline (PBS) in order to obtain a concentration of [100 lM] for each fraction. The aggregation of Ab(35– 25) and Ab(25–35) was carried out via a 36-h incubation at 37 °C (Limo´n et al., 2009a,b). Stereotaxic surgery Each animal was anesthetized with ketamine-xylazine (75:10 mg/kg, ip) and then randomly assigned to the experimental groups: vehicle; Ab(35–25); ACEA; AM-251; Ab(25–35); ACEA + Ab(25–35); AM-251 + Ab(25–35); and, AM-251 + ACEA +Ab(25–35) (n = 12 per group). Each animal was placed in a stereotaxic apparatus (Stoelting Co., Wood Dale, IL, USA) for the administration of vehicle, Ab(35–25), ACEA, AM-251, or Ab(25–35). Each solution was injected using a 10-lL Hamilton syringe (Nanomite Syringe Pump, Harvard Apparatus), with each 1 lL of solution infused for 300 s. 1 lL of ACEA (1 lM) or 1 lL of AM-251 (10 lM) was administrated, after which 1 lL of Ab(25–35) (100 lM) peptide was bilaterally injected into the CA1 subfield of the hippocampus, with a 10-min interval between the injection of each agent at the following stereotaxic coordinates: AP: 3.8 mm from the Bregma; L: ±3.0 mm from the midline; and, DV: 2.0 below the dura (according to Paxinos and Watson’s Stereotaxic Atlas, 1998). The vehicle group was administered with 1 lL of DMSO + 1 lL of PBS, while the Ab(35–25) was administered with 1 lL of Ab(35–25) (100 lM) + 1 lL of PBS, both into the CA1 subfield of the hippocampus. Proper post-operative attention was provided until the animal made a full recovery. Behavioral testing The time sequences for the behavioral tests are shown in Fig. 1. To evaluate the learning and memory process, the

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Fig. 1. Scheme of the experimental sequence. Eight groups were obtained: vehicle; Ab(35–25); ACEA; AM-251; Ab(25–35); ACEA +Ab(25–35); AM251 + Ab(25–35); and, AM-251 + ACEA +Ab(25–35). The cognitive assessment was conducted on the 20th day for learning and on the 29th day for memory in the eight-arm radial maze. Finally, the nitric oxide, iNOS, GFAP and Iba-1markers levels and cellular damage were observed using Fluoro-Jade B in the hippocampus of the rats.

eight-arm radial maze was used according to the protocol previously reported by Patricio-Martı´ nez et al. (2016). Spatial learning was conducted during the first 2 days of testing in the radial arm maze (20–21 days post-Ab(25– 35) injection) over the course of 20 trials for a 2-day period (10 trials per day). In each trial, the animals were placed on a central platform located at the middle of the eightarm radial maze and allowed to move freely around the maze until the animal had either completed the number of permitted attempts or the allotted time period (200 s) had elapsed. During the entire test, only three of the eight arms contained pellets. While, in the first three trials, each rat was permitted to choose eight arms, the number of permitted attempts was reduced in the later trials, so that, by the second day of training, the rat was permitted to choose only three arms for each trial, after which the animal was removed from the maze. Spatial learning was evaluated using the percentage of correct responses and the number of reference errors, as previously reported (Limo´n et al., 2011). The percentage of correct responses was defined as the number of entries into arms containing pellets, divided by the number of entries into all arms and then multiplied by 100. The number of reference errors was defined as the number of entries, in each trial, into arms that did not contain pellets. The memory test was carried out 8 days after the learning test, and comprised one trial, in which the animal made three test choices of entries into the arms either with or without pellets before the time elapsed (200 s). The memory test assessed the percentage of correct responses using the same method used during the learning test and the latency to the third correct response in seconds, in order to evaluate the time the animals required to remember where the pellet was. Nitrite assay The animals were decapitated after the memory test (n = 6 per group), with their brains then immediately removed and washed in ice-cold SSI. The hippocampal region and frontal cortex were then dissected and

homogenized in 3 mL of ice-cold 0.1 M PBS, pH = 7.4. The homogenate was centrifuged at 12,500 rpm (4 °C), with the supernatant obtained then stored at 70 °C. The supernatants were used for protein and nitrite level measurements, with nitrite levels measured using the Griess method (Green et al., 1982). The Griess reagents were sulfanilamide, glacial acetic acid, and N-(1-naphthyl) ethylenediamine (Sigma, St. Louis, MO, USA), with 50 lL of the Griess reagent added directly to a 50-lL aliquot of the homogenate and incubated under reduced light for 5 min. Samples were analyzed at 540 nm in an ELISA microplate reader. The protein level of each sample was determined using the Bradford method, where 20 lL of the supernatant was taken, to which 180 lL of the Bradford reagent was added. Data were calculated as lM of nitrite per mg of protein contained in 50 lL of the sample. Histological examination After the behavioral experiments, all animals (n = 6 per group) were anesthetized and intracardially perfused with 200–250 mL of 4% paraformaldehyde in phosphate-buffered saline (PBS, pH 7.4). The brains were removed and embedded in paraffin, with 5-lm coronal brain sections then obtained for histological analysis using a microtome (Leica RM2125RT) at the level of the hippocampus, approximately 2.8 to 4.3 from the bregma. Immunohistochemistry The paraffin was removed section-by-section, which were then rehydrated using conventional histological techniques and rinsed with PBS pH 7.4. Non-specific binding sites were blocked by means of incubation in IgG-free 2% bovine serum albumin (BSA, Sigma) for 60 min. Specimens were then incubated for 10 min with 0.2% Triton X-100 in PBS at room temperature, with the slices then rinsed with PBS. The sections were incubated overnight at 4 °C in the corresponding antibody: polyclonal rabbit antibody anti-inducible nitric oxide synthase (iNOS) (sc-651, Santa Cruz Biotechnology

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Inc.), diluted to 1:100; polyclonal rabbit antibody anti-Glial fibrillary acidic protein (GFAP), diluted to 1:1000 (Dako, Denmark A/S); or, monoclonal rabbit antibody antiIonized calcium binding adaptor molecule 1 (Iba-1), diluted to 1/500 (Wako Pure Chemical Industries, Ltd. Osaka, Japan). Primary (rabbit) antibody labeling was recognized using a secondary FITC (iNOS and Iba-1) and visualized in the green channel, while primary antibody was recognized using a secondary conjugated rhodamine (GFAP) and visualized in the green channel. The immune-stained neurons in the CA1 subfields of the hippocampus were observed through a Leica DM/LS fluorescent microscope at 40 (Leica DM/LS Microsystems, Wetzlar, GmBH), while the photos were taken using a Leica DFC-300FX digital camera (Leica Microsystems Digital Imaging, Coldhams Lane, Cambridge, UK). The sections used were taken close to the injection site for qualitative assessment. The photographs, taken by fluorescence microscope at 40  average in the CA1 region of the hippocampus, were used to quantify iNOS, Iba-1 and GFAP immunoreactivity. The digitized images were transformed into TIFF files both for storage and to facilitate subsequent analysis. For Iba-1 and GFP, solely the percentage of the total area of the photomicrograph that had fluorescent staining was analyzed, with the threshold then chosen from the vehicle group and applied to all experimental groups. The iNOS immunoreactivity images were converted to gray scale for the quantification of neurons using the Image J software. Fluoro-Jade B stain The paraffin-embedded sections (5 lm) were rehydrated using conventional histological techniques and rinsed with PBS, pH 7.4. The slides were then placed in a 0.06% potassium permanganate solution for 10 min, washed in distilled water for 2 min, and then rinsed in 0.006% Fluoro-Jade B (Chemicon, International Inc. USA) prepared in 0.1% acetic acid. After 20 min, the slides were washed for 1 min in distilled water, cleared with xylene and finally mounted. Statistics The results were expressed as mean ± standard error of the mean (S.E.M.). The results of the spatial learning and memory tests, the nitrite levels and the data obtained from the image analysis of the iNOS, Iba-1 and GFAP and Fluoro-Jade B in the CA1 subfields of the hippocampus were subject to a one-way analysis of variance (ANOVA). The relevant differences were analyzed pairwise by means of post-hoc comparisons conducted using a Tukey test to determine specific group differences. The criterion for significance was P < 0.05.

RESULTS The acute activation of CB1r in the hippocampus prevents cognitive impairment by Ab(25–35) To understand the protective effect of the activation of CB1r by ACEA on the cognitive processes of animals

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injected with Ab(25–35) into the hippocampus, spatial learning and memory were tested using the eight armradial maze (Fig. 1). The spatial learning test showed normal acquisition levels for information processing in the animals from the vehicle group, which recorded 57% of correct responses and minimum values for the number of reference errors (mean 1.2). In order to ascertain the effect of non-toxic Ab(35–25)-peptide on learning and memory, an Ab(35–25)-treated group performed the test. The Ab(35–25) group presented 56% correct responses and low number of reference errors (mean 1.3), indicating that the Ab(35–25) group followed a similar learning process to the vehicle group. The ACEA and AM-251 groups recorded 61% and 60% correct responses, respectively, and minimum values for the number of reference errors (mean 1.1 and 1.2, respectively) (Fig. 2 A and B), indicating that the ACEA and AM-251 groups followed a similar learning process to the vehicle group. The Ab(25–35) group presented 44% correct responses, as well as a greater number of reference errors (mean 2.2) during the spatial learning test (P < 0.05). In contrast, the ACEA plus Ab(25–35) group did not show a spatial learning deficit, in that these animals recorded 61% correct responses with a minimum number of reference errors (mean 1.1). Therefore, the ACEA treatment provides protection against learning impairment, at a level of 30% (p < 0.01). On the other hand, the AM-251 plus Ab(25– 35) group and the AM-251 plus ACEA plus Ab(25–35) group did not experience improvements in their learning process, with both groups presenting similar behavior to the Ab(25–35) group, which presented 47% and 41% correct responses, respectively, and a higher number of reference errors (mean 2.2 and 2.1 number of reference errors, respectively). In the findings for memory processes, the Ab(25–35) group presented a spatial memory deficit (mean of 30% correct responses) (Fig. 2 C), compared with the vehicle group (p < 0.05), which recorded 64% correct responses. The Ab(35–25) group recorded 67% correct responses during the spatial memory test, with no statistical differences found between this group and the vehicle group. During the spatial memory test, the ACEA and AM-251 groups presented 67% and 61% correct responses, respectively, with no statistical differences found between them and the vehicle group. Moreover, the ACEA plus Ab(25–35)-treatment group presented 70% correct responses (p < 0.01), indicating that these animals did not lose their spatial memory and achieved a similar performance level as the vehicle group in the memory test. The spatial memory of the AM-251 plus Ab(25–35) group and AM-251 plus ACEA plus Ab(25–35) group did not improve in terms of their memory process, as both groups presented similar behavior to the Ab(25–35) group, which presented 36% and 33% correct responses, respectively. Finally, the latency to the third correct response in the memory process was evaluated, in which the vehicle group took 138 s to perform the task, the Ab(35–25) group took 146 s, with the ACEA and AM-251 groups taking 143 and 145 s, a similar time period, respectively. The Ab(25–

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Fig. 2. The activation of CB1 receptor by ACEA prevents the loss of spatial learning and memory caused by Ab(25–35). Spatial learning was tested in the eight-arm radial maze on the 20th and 21st days after ACEA + Ab(25–35) injection. (A) Spatial learning was evaluated using the percentage of correct responses and (B) the number of reference errors. Spatial memory was tested in the eight-arm radial maze on the 29th day after ACEA +Ab(25–35) injection. (C) Spatial memory was evaluated using the percentage of correct responses, as was (D) the latency to the third correct response (s). Data show the mean values ± SEM. Statistics were determined via one-way ANOVA, followed by the application of a Tukey’s test for post hoc analysis: *P < 0.05, ***P < 0.001 vs vehicle, Ab(35–25), ACEA and AM-251 groups; #P < 0.05, ##P < 0.01, ###P < 0.001 vs Ab(25–35) group and &P < 0.01, &&&P < 0.001 vs ACEA + Ab(25–35) group. 35) group increased its latency, performing the task in 173 s (p < 0.001). In contrast, the latency of the ACEA plus Ab(25–35) group decreased for the third correct response (mean 128 s) (p < 0.001) (Fig. 2 D), while neither the AM-251 plus Ab(25–35) group nor the AM-251 plus ACEA plus Ab(25–35) group improved in terms of latency (mean 177 and 172 s, respectively), thus behaving similarly to the Ab(25–35) group. These results show that ACEA protects against the loss of memory caused by Ab(25–35) toxicity in the hippocampus.

The administration of ACEA in the CA1 of the hippocampus prevents the NO production and iNOS expression caused by Ab(25–35) toxicity The release of NO was evaluated by measuring nitrites (NO2 ), a stable metabolite of NO, in both the hippocampus and frontal cortex. The results presented here show that 29 days after the Ab(25–35) injection, the nitrite levels increased by a mean 9.8 lM/mg of protein (p < 0.05) compared to a mean 3.8 lM/mg and a 5.0 lM/mg mean of protein for the vehicle and Ab(35–25)

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Fig. 3. ACEA-attenuated Ab(25–35)-induced increases in nitric oxide levels in the hippocampus. Nitrite levels (NO2 ) were quantified in the hippocampus (A) and frontal cortex (B) n = 6 per group. The values show the mean of nitrites [lM]/mg of protein ± SEM. Statistics were determined with a one-way ANOVA test, followed by the application of a Tukey’s test for post hoc analysis: **P < 0.01 and *P < 0.05 vs vehicle, ACEA and AM-251 groups and #P < 0.05 vs Ab(25–35) group.

groups respectively (Fig. 3A). Moreover, the groups treated with ACEA and AM-251 presented 3.7 and 4.4 lM/mg of protein in their nitrite levels, results which did not reveal statistically significant differences to the results obtained for the vehicle group. In contrast, the nitrite levels for the ACEA plus Ab(25–35)-treated group were mean 4.8 lM/mg of protein (p < 0.05) lower than the levels found for the Ab(25–35) group. The nitrite levels for the AM-251 plus Ab(25–35) and AM-251 plus ACEA plus Ab(25–35) groups, however, were not lower (mean 8.2 and 9.0 lM/mg of protein), with both groups presenting similar behavior to the Ab(25–35) group. Finally, when assessing the nitrite levels in the frontal cortex, no statistically significant difference was found for any of the experimental groups (Fig. 3B). As the enzyme iNOS is involved in the synthesis of NO and, given the increased nitrite levels found in the hippocampus, the expression of iNOS in the CA1 subfields of the hippocampus was studied in the present research (Fig. 4). The results show that Ab(25–35) caused a significant increase in the number of iNOS-expressing cells (mean 38.3) (p < 0.001; p < 0.001; p < 0.01) in the CA1 subfields of the hippocampus compared to the vehicle (mean 4.7), Ab(35–25) (mean 4.0), ACEA (mean 2.0) and AM-251 (mean 2.5) groups. However, the number of iNOS- expressing cells (mean 16.0) (p < 0.05) decreased for the ACEA plus Ab(25–35)treated group compared with the Ab(25–35) treatment group, while the number of iNOS-expressing cells (mean 50.7 and 56.0, respectively) for the AM-251 plus Ab(25–35) group and AM-251 plus ACEA plus Ab(25–35) group did not decrease, indicating behavior similar to that presented by the Ab(25–35) group (Fig. 4 A).

The findings of this study suggest that ACEA prevents increased nitrite levels and iNOS expression in the hippocampus. The acute activation of CB1r by ACEA decreases gliosis in rats as caused by Ab(25–35) injury The activation of the glial cells is crucial during the neuroinflammatory process. Specifically, astrocytes (GFAP protein) and microglia (Iba-1 protein) are important cellular groups that, upon over-activation, increase the expression of iNOS and, consequently, NO production. The present study evaluated the protective effect of the acute activation of CB1r against the gliosis process induced by Ab(25–35). The results show that 29 days after Ab(25–35) injection, the levels of GFAP and Iba-1 in the CA1 subfields of the hippocampus increased by 10.3% and 2.5%, respectively, while the vehicle and Ab(35–25) groups presented 6.7% and 5.3% of the stained area for GFAP, respectively, and 1.7% and 1.6% of the stained area for Iba-1) (P < 0.01). The ACEA and AM-251 groups show stained area percentages (5.0% and 5.4 of the stained area for GFAP, and 1.4% and 1.6 of the stained area for Iba-1) similar to the control group. In contrast, the ACEA plus Ab(25–35)-treated group presented 7.3% (P < 0.05) and 2.4% (P < 0.01) of the stained area in the hippocampus for GFAP and Iba-1, respectively. Furthermore, while the groups treated with AM-251 plus Ab(25–35) and AM-251 plus ACEA plus Ab(25–35) did not record a significant GFAP increase, they did record a significant increase in Iba-1 (2.4% and 2.3% of the stained area) (P < 0.01) in the CA1 subfields of the hippocampus (Fig. 5 A and B).

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Fig. 4. ACEA decreases the iNOS expression induced by Ab(25–35) in the hippocampus.Photomicrographs of the CA1 region of the hippocampus section, stained for iNOS (green) for each experimental group. (A) Histograms show the proportion iNOS+ cells in the CA1 region of the hippocampus n = 6 per group. Bar indicates 50 lm. The values show mean ± SEM, with statistics determined via a one-way ANOVA test, followed by the application of a Tukey’s test for post hoc analysis: ***P < 0.001 vs vehicle, Ab(35–25), ACEA and AM-251 groups; #P < 0.05 vs Ab(25–35) group and &&&P < 0.001 vs ACEA + Ab(25–35) group. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

The immunoreactivity of GFAP and Iba-1 in the dentate gyrus (DG) (Fig. 6) was also analyzed. The results found that Ab(25–35) caused a significant increase, 10.7% and 3.5% of the stained area of the DG, for GFAP and Iba-1 (P < 0.01, P < 0.001), respectively, compared to the vehicle and Ab(35–25) groups (5.3% and 5.9% of the stained area for GFAP and 2.0% and 2.1% of the stained area for Iba-1). The ACEA and AM-251 groups show similar stained area percentages (5.4% and 6.7% of the stained area for GFAP, and 2.3% and 2.4% of the stained area for Iba-1) to the control group. Moreover, it was observed that ACEA plus Ab(25–35) prevents the increase of GFAP and Iba-1 in the DG (6.1% of the stained area for GFAP and 2.1% of the stained area for Iba-1) (P < 0.05, P < 0.001) (Fig. 6 A and B). Although the group treated with AM-251 plus Ab(25–35) presented a higher percentage of stained area for GFAP in the DG (10.7%), this was not the case for Iba-1. The AM-251 plus ACEA plus Ab(25–35) group recorded a significant increase in GFAP and Iba-1 (11.5% and

4.1% of the stained area for both enzymes) (P < 0.05, P < 0.001) in the DG. The findings of this study show that the acute activation of CB1r by ACEA prevents increased GFAP and Iba-1 levels in the CA1 and DG of the hippocampus. The acute activation of CB1r via ACEA administration in the CA1 of the hippocampus provides neuronal protection against Ab(25–35) injury To further confirm the protective effect of the CB1r agonism via activation by ACEA against the toxicity induced by the Ab(25–35) peptide, this study evaluated neurodegeneration in the CA1 subfields of the hippocampus using Fluoro-Jade B staining (Fig. 7). Fluoro-Jade B stains bodies, axons and degenerate dendritic terminals without staining healthy neurons (Schmued and Hopkins, 2000; Ortega et al., 2014). The injection of the Ab(25–35) peptide revealed neurons stained with Fluoro-Jade B in the CA1 subfields of the

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Fig. 5. ACEA decreases the GFAP expression induced by Ab(25–35) in the CA1 subfields of the hippocampus.Photomicrographs of the CA1 region of the hippocampus section, stained for GFAP (red, above) and Iba-1(green, below) for each experimental group. (A) Scheme of CA1-Hp localization in a rat brain coronal slide and histograms show the proportional stained area of GFAP-ir and Iba-1-ir (B) in the CA1-Hp n = 6 per group. Bar indicates 50 lm. The values show mean ± SEM, with statistics determined with a one-way ANOVA test, followed by the application of a Tukey’s test for post hoc analysis: **P < 0.01 vs vehicle and #P < 0.05 vs Ab(25–35) group. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

hippocampus, with the quantitative analysis showing that the Ab(25–35) group presented 0.9% stained area, while the control group presented 0.05% of the stained area for fluoro-Jade B. The ACEA and AM251 groups presented a low percentage of stained area (0.06% and 0.02%, respectively) for fluoro-Jade B, which is indicative of an absence of neurodegeneration. While the AM251 plus Ab(25–35) and AM251 plus ACEA plus Ab(25–35) groups showed degenerating neurons, this was in a smaller proportion than the group administered with the Ab(25–35) peptide, corresponding to 0.32% and 0.46, respectively, of the stained area for fluoro-Jade B (Fig. 7 A). These results suggest that the activation of CB1r by ACEA decreases the number of neurons that undergo the death process.

DISCUSSION The present study shows specific agonist of CB1r AD using Ab(25–35). intrahippocampal CB1r performance of animals

the neuroprotective effect of a in a non-transgenic model of The acute activation of is shown to improve the in the learning and spatial

memory test conducted in the eight-arm radial maze, thus suggesting that CB1rs are involved in learning and memory processes in the hippocampus. In addition, at a cellular level, the activation of CB1r is able to decrease the protein expression of iNOS and, consequently, the production of NO, which results in a reduced glial response and, thus, a retarding of the neurodegenerative process. The toxicity mechanisms of Ab(25–35) are involved in the modification of intracellular Ca2+ concentrations via the activation of the NMDA receptor (Chen et al., 2007; Resende et al., 2007), mitochondrial dysfunction (Casley et al., 2002), the production of ROS, and inflammation, which induces neurodegeneration (Limo´n et al., 2009b; Dı´ az et al., 2012). The activation of CB1r, which is coupled to an inhibitory G protein (Gi), promotes the inhibition of adenylate cyclase (AC), inducing a decrease in the level of second messengers such as cAMP, an event related to a decrease in the release of glutamate (Sagan et al., 1999; Di Marzo et al., 2000; Breivogel et al., 2001; Vogel et al., 1993; Howlett, 1995; Howlett et al., 2002). The decreased glutamate release may be one of the mechanisms by which the administration of ACEA protects the excitotoxicity of the

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Fig. 6. ACEA decreases GFAP and Iba-1 expression induced by Ab(25–35) in the DG.Photomicrographs of the DG section stained for GFAP (red, above) and Iba-1(green, below) for each experimental group. (A) Scheme of the DG localization in a rat brain coronal slide and histograms show the proportional stained area of GFAP-ir and Iba-1-ir (B) in the CA1-Hp n = 6 per group. Bar indicates 50 lm. The values show mean ± SEM, with statistics determined via a one-way ANOVA test, followed by the application of a Tukey’s test for post hoc analysis: **P < 0.01, ***P < 0.001 vs vehicle; #P < 0.05, ###P < 0.001 vs Ab(25–35) group and &P < 0.05, &&&P < 0.001 vs ACEA +Ab(25–35) group. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Ab(25–35) peptide, thus ensuring correct cellular communication and the integration of neuronal circuits, a process which improves learning and, consequently, spatial memory. Several reports mention that the continuous systemic and intracranial administration of cannabinoid agonists induces deficits in hippocampal-dependent tasks carried out in the radial maze, the Morris water maze and the T-maze (Lichtman et al., 1995, Ferrari et al., 1999; Da Silva and Takahashi, 2002; Egashira et al., 2002; Suenaga and Ichitani, 2004). In contrast, CB1r antagonists show higher performance in hippocampaldependent memory tasks (Terranova et al., 1996). Interestingly, we have shown that the administration of ACEA and AM-251 alone does not cause changes in the acquisition and retrieval of information in the eight-arm radial maze, results indicating that the doses of each drug administered here correspond to non-amnesic doses. It is important to note that, as demonstrated in the present study, the scenario changes when cannabinoid agonists are used in animals subject to toxic insult, as they trigger mechanisms involved in both neuronal protection and protection against excitotoxicity and neurodegeneration

(Marsicano et al., 2003; Lastres-Becker et al., 2005; Aguado et al., 2007). On the other hand, the AM-251 plus Ab(25–35) and AM-251 plus ACEA plus Ab(25–35) groups did not present improved learning and memory, an effect which may be due to the action of the antagonist AM251, which, once bound to the CB1r, is unable to activate the signaling involved in diminishing the excitotoxic effect induced by Ab(25–35). Moreover, when bound to CB1r, AM251 will prevent the binding of ACEA, given that the receptor is occupied; therefore, the effect observed on learning and memory corresponds to the mechanism affected by the Ab(25–35) peptide, thus corroborating that the protective effect observed in the ACEA plus Ab(25– 35) group corresponds to the activation of CB1r. Additionally, the effects observed of the nontoxic-Ab(35–25) fraction on learning and memory show that this peptide did not cause cognitive deficits compared to the results obtained for the vehicle group, meaning that only the Ab(25–35) group presents the neurotoxic properties of the amyloid beta peptide. Our research group has studied the cellular mechanisms by which the Ab(25–35) peptide could

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Fig. 7. ACEA prevented Ab(25–35)-induced increases in neurodegeneration in the hippocampus.Photomicrographs of the Fluoro-Jade Bstained section of the CA1 subfield of the hippocampus show increased staining, thus indicating Ab(25–35)-induced neurodegeneration, in contrast to the ACEA plus Ab(25–35) group, which presented a lower level of neurodegeneration. (A) Histograms show the proportional stained area of FluoroJade-ir in the CA1-Hp n = 6 per group. Bar indicates 50 lm. The values show mean ± SEM, with statistics determined with a one-way ANOVA test, followed by the application of a Tukey’s test for post hoc analysis: **P < 0.01, ***P < 0.001 vs vehicle, ACEA, AM-251; ##P < 0.01 vs Ab(25–35) group. Bar indicates 50 lm.

promote deterioration in learning and memory. Among the different pro-inflammatory proteins produced in response to the oxidative stress induced by Ab(25–35) are inducible nitric oxide synthase (iNOS) and its enzymatic product, nitric oxide (NO) (Limo´n et al., 2009a; Dı´ az et al., 2010, 2011, 2012), markers which are considered the most important neurotoxic effectors in AD (Togo et al., 2011). The participation of NO in the neurodegenerative process induced by Ab(25–35) has been demonstrated by inhibiting NOS (iNOS and nNOS) activity, resulting in decreased nitrite (a stable metabolite of NO) concentration and improving learning and spatial memory (Dı´ az et al., 2011, 2014). nNOS and eNOS have been described as constitutively expressed isoforms, which are activated by an increase in intracellular Ca2+; however, iNOS promotes an increase in the synthesis of NO in response to the inflammatory process. The results of the present study show that CB1r agonism via ACEA, in the presence of the Ab(25–35) peptide, prevents the production of NO and, consequently, causes a decrease in the amount of iNOS enzyme in the hippocampus. It is important to mention that the production of NO observed in the present study is a consequence of the activity of all NOS isoforms, although iNOS has a fundamental role in glia cell activity and was the enzyme studied in the present research. Therefore, the present study has demonstrated that the acute activation of CB1r is capable of decreasing the level of the iNOS enzyme and, consequently, the production of NO. Esposito et al. (2006) found that, in PC12 cells exposed to the Ab peptide, cannabidiol (CBD) decreases the expression of iNOS, NO and NF-jb factor (the transcription factor promoting

iNOS expression). This finding concurs with the results of the present study, which show that an acute activation, via ACEA, of CB1r in rats treated with the Ab(25–35) peptide decreases the level of iNOS and NO expression. The effect of ACEA was reversed by the antagonist AM-251, with the AM-251plus Ab(25–35) and AM-251 plus ACEA plus Ab(25–35) groups showing no decrease in either NO concentration or the number of cells positive to iNOS at a hippocampus level. These results are due to the fact that AM-251 antagonizes the CB1r, with the observed effect resulting from the neurotoxic effect induced by Ab(25–35), thus proving, once again, that the protective effect observed is the result of the acute activation of CB1r. The control groups did not show increases in NO levels or the protein expression of iNOS, which was indicative of the absence of cellular stress. Evaluations of NO concentrations in the frontal cortex revealed no significant changes in the experimental groups. Although NO is a highly diffusible gas, no evidence was found that the intrahippocampal injection of Ab(25–35) had an impact on another brain area such as the frontal cortex. For this reason, the present research only evaluated markers of neuroinflammation and neurodegeneration in the hippocampus. There is evidence that the expression of the iNOS protein increases in patients with AD; furthermore, this over-expression has also been shown to play an important role in both the inflammatory and neurodegenerative processes that occur in both AD in humans and animal models (Togo et al., 2011; Limo´n et al., 2009a; Dı´ az et al., 2012).

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Several studies have shown that the injection of the Ab(25–35) peptide promotes the inflammatory process through the activation of glial cells (astrocytes and microglia) (Combs et al., 2001; Ramirez et al., 2005), in turn causing the astrocytes and microglia to increase the expression of iNOS, and promoting high concentrations of NO and proinflammatory cytokines, such as IL1b, IL-6 and TNFa, which are key molecules in the neurotoxicity induced by the administration of Ab(25–35) (Stepanichev et al., 2008; Dı´ az et al., 2011, 2012; Zussy et al., 2013). The main features of reactive gliosis are hypertrophy and astrocytic proliferation, together with the overexpression of intermediate filament markers such as the GFAP protein, the most well-known label of active astrocytes. The present study found that the administration of the Ab(25–35) peptide increases the expression of GFAP and Iba-1 proteins, which suggests an inflammatory environment that contributes to neuronal damage. The results of the present study show that, in the CA1 region of the hippocampus, while ACEA treatment in rats lesioned with Ab(25–35) decreases the expression of GFAP, it does not decrease Iba-1 expression in the same region; however, in the DG region, the treatment is found to cause a decrease in the expression of both markers. This may be due to the fact that the CB1rs are found in greater proportion in cells of the astrocytes (Gu tie´rrez-Rodrı´ guez et al., 2018), while the CB2rs are mostly found in the cells of the microglia (Lou et al., 2011). Although CB2r is broadly related to the modulation of the inflammatory response, the results presented here show that CB1r activation is capable of decreasing the expression of GFAP protein. The low level effect of ACEA on the expression of Iba-1 may be due to CB2r being largely confined to microglial cells (Nun˜ez et al., 2004). Therefore, the use of a non-selective CB/CB2r agonist could better decrease the expression of microglia and, consequently, neuroinflammation. We found the same behavior pattern in the AM-251 plus Ab(25–35) and AM251 plus ACEA plus Ab(25–35) groups, as they do not show a decrease in GFAP and Iba markers, data which relate to prior results in that these treatments do not decrease the expression of iNOS and NO, nor do they improve the memory process, a consequence of the antagonism of CB1r. Interestingly, we found that the Ab(35–25), AM-251, and ACEA groups did not modify the expression of GFAP and Iba proteins, a finding related to the absence of cellular stress markers such as NO and iNOS. On evaluating the neurodegenerative process induced by Ab(25–35) in the CA1 region of the hippocampus, the present study found that the ACEA plus Ab(25–35) group did not undergo neurodegeneration, in contrast with the Ab(25–35) group. It has been shown that cannabinoids have an important role in neurodegenerative processes, with experiments conducted on animals, in whom CB1r has been eliminated, showing that they are more susceptible to neurodegeneration (Parmentier-Batteur et al., 2002; Marsicano et al., 2003). On the other hand, CB1r knockout mice presented a decrease in BDNF expression (Aso et al., 2008) with the induction of BDNF expression contributing to the neuroprotective effect of CB1r, which

strongly suggests that BDNF is a key mediator in CB1rdependent protection against excitotoxicity (Marsicano et al., 2003; Khaspekov et al., 2004). This finding reinforces the hypothesis that CB1r may be a potential therapeutic target in the treatment of neurodegenerative processes. The present study shows that the acute activation of CB1r is crucial to reducing neurodegeneration, probably due to a decrease in the release of glutamate caused by the reduction of the amount of calcium and the inhibition of signaling pathways harmful to neurons, such as the iNOS signaling pathway and that involved in the synthesis of NO. Other studies have shown that cannabinoids prevent the occurrence of Ab toxicity in PC12 cells by reducing the production of reactive species, lipid peroxidation and caspase-3 levels (Iuvone et al., 2004). As expected, no diminishment was found in the neurodegenerative processes observed in the Ab(25–35) plus AM-252 and Ab(25–35) plus AM-251 plus ACEA groups, which is due to the blockade of CB1r and the damage induced by the Ab(25–35) peptide. The presence of neurodegeneration correlates with the increase in the expression of iNOS, the increase of NO, the activation of glial cells and decreased memory. Furthermore, no neurodegenerative processes were seen to change in animals treated with solely ACEA, AM-2-51 or Ab(35–25), findings which indicate that only the neurotoxic Ab(25–35) fraction is able to cause neuronal death in the hippocampus. Collectively, all data suggest that ACEA protects against the neurodegeneration caused by Ab(25–35). In summary, the present study shows that the acute activation of CB1r prevents memory loss in rats lesioned with Ab(25–35). These results can be explained by the activation of CB1r, which prevents iNOS expression and consequently decreases NO production, thus preventing neurodegeneration in the CA1 region of the hippocampus. These findings suggest that the early activation of the CB1r is crucial to neuroprotection.

ACKNOWLEDGMENTS Thanks to Benjamin Stewart (English language native and academic proof-reader) for editing the English language text.

FUNDING This study was supported by grants from VIEP-BUAP2018-2019 given to ID. Limo´n.

AUTHOR CONTRIBUTIONS APM and RSZ contributed equally to this study and both are considered first authors. APM, IAC, LGP and RSZ performed the experiments. APM, RSZ and IDL wrote or contributed to the writing of the manuscript. APM and IDL were responsible for the conception, analysis and interpretation of the data. IDL approved the final version for publication.

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DECLARATION OF COMPETING INTEREST All authors of this article declare no conflict of interest.

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(Received 21 February 2019, Accepted 1 August 2019) (Available online 7 August 2019)