Photostimulation of Locus Coeruleus CA1 catecholaminergic terminals reversed spatial memory impairment in an Alzheimer's disease mouse model.

Photostimulation of Locus Coeruleus CA1 catecholaminergic terminals reversed spatial memory impairment in an Alzheimer's disease mouse model. | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Photostimulation of Locus Coeruleus CA1 catecholaminergic terminals reversed spatial memory impairment in an Alzheimer's disease mouse model. Donovan K. Gálvez-Márquez, Oscar Urrego-Morales, Luis F. Rodríguez-Durán, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5868268/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Rationale One of the earliest changes associated with Alzheimer's disease (AD) is the loss of catecholaminergic terminals in the cortex and hippocampus originating from the Locus Coeruleus (LC). This decline leads to reduced catecholaminergic neurotransmitters in the hippocampus, affecting synaptic plasticity and spatial memory. However, it is unclear whether restoring catecholaminergic transmission in the terminals from the LC may alleviate the spatial memory deficits associated with AD. Objectives This study investigates how optogenetic stimulation of catecholaminergic projections from the locus coeruleus to the hippocampal CA1 region may enhance spatial memory and alleviate synaptic plasticity deficits associated with Alzheimer's disease. Methods We conducted experiments using a 12-month-old 3xTg-AD mouse model (AD-TH), which expresses Cre recombinase under the control of the tyrosine hydroxylase (TH) gene. This model allowed us to photostimulate the terminals from the locus coeruleus in the hippocampal CA1 region before performing two different spatial memory tasks and inducing long-term potentiation (LTP). Results Optogenetic stimulation successfully reversed the impairment of spatial memory retrieval in aging AD-TH mice. Furthermore, this stimulation restored catecholaminergic neurotransmitter levels in the hippocampus and enhanced synaptic plasticity, as demonstrated by an LTP protocol. Conclusions These findings indicate that the catecholaminergic circuitry from the locus coeruleus (LC) to the hippocampal CA1 region plays a crucial role in disrupting synaptic plasticity and contributing to the spatial memory deficits seen in the early stages of AD. This study highlights the potential therapeutic benefits of targeting LC catecholaminergic neurons to improve cognitive function in patients with AD. Cognitive Neuroscience Neurobiology of Disease Dopamine Alzheimer`s disease Locus Coeruleus Hippocampus Cognitive impairment Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Alzheimer's disease (AD) is characterized by the accumulation of amyloid beta (Aβ) peptides in extracellular aggregates and the formation of intracellular neurofibrillary tangles composed of Tau protein (O’Brien and Wong 2011; Sadigh-Eteghad et al. 2015; Zhang et al. 2018; Hampel et al. 2021). The build-up of Aβ peptide and hyperphosphorylated Tau protein tangles in different brain parts has been shown to disrupt the neurotransmitter system, synaptic plasticity, and cognitive function in laboratory and clinical studies (Tolar et al. 2021; Takahashi et al. 2021; Gloria et al. 2021; Wang et al. 2024). However, despite being the leading indicator of this neurodegenerative disease, a direct mechanism explaining the cognitive decline has not been established (Ratan et al. 2023). Despite numerous attempts to address these pathological hallmarks through clinical approaches, developing effective treatments for AD remains challenging (Kuo and Rajesh 2019; Passeri et al. 2022). This emphasizes the urgent need for a deeper understanding of the mechanisms behind the cognitive decline in AD (Ratan et al. 2023). The hippocampus plays a crucial role in processing spatial memory and is significantly affected in AD patients from the early stages (Philippen et al. 2024; Billaud and Yu 2024). Initial investigations of post-mortem AD brains have revealed significant disturbances in the hippocampal structure and connectivity, including catecholaminergic projections from the Ventral Tegmental Area (VTA) and Locus Coeruleus (LC) (Sala et al. 2021; Dai et al. 2023). Additionally, AD murine models show diminished catecholaminergic innervation within the hippocampus, correlating with alterations in neuronal excitability and modified gamma-wave oscillatory activity (Nobili et al. 2017; Sakai et al. 2023; Spoleti et al. 2024). Disturbances of the catecholaminergic system, such as decreased catecholamine neurotransmitter levels (Pan et al. 2020) and quantities of catecholamine receptors (Kemppainen et al. 2003; Szot et al. 2006), are common in AD. Damage to catecholaminergic neurons has been linked to memory deficits in animal models of AD and may contribute to the initial symptoms experienced by AD patients (Gloria et al. 2021; Iannitelli et al. 2023; Bueichekú et al. 2024). Moreover, several studies have suggested the crucial role of catecholaminergic transmission in improving memory deficits in AD. Pharmacological activation of the catecholaminergic system through the use of dopamine (DA) agonists, the DA precursor L-DOPA, or the monoamine oxidase-B inhibitor selegiline has been shown to enhance synaptic plasticity, increase dendritic spine density, modulate hippocampal postsynaptic composition, and improve memory deficits in experimental AD models (Swanson-Park et al. 1999; Yuan Xiang et al. 2016; Moreno-Castilla et al. 2016; Tábi et al. 2020; Tsetsenis et al. 2022; Alborghetti et al. 2024; Basir et al. 2024). Recent research has shifted focus from the VTA to the LC as a significant catecholaminergic neuromodulatory structure in the hippocampal circuitry for memory modulation. The LC, which sends denser catecholaminergic inputs to CA1 than the VTA, has been found to modulate synaptic plasticity and spatial memory processes (Takeuchi et al. 2016; James et al. 2021; Gálvez-Márquez et al. 2022). In the early stages of AD, damage and degeneration of the catecholaminergic neurons emerge as a direct consequence of high energy expenditure and large oscillations on intracellular Ca2+, leading to oxidative stress. Furthermore, the DA can be auto oxidized, producing dopaquinone and reactive oxygen species. This oxidative stress increases proinflammatory cytokine expression, disrupting synaptic efficiency (Tönnies and Trushina 2017). Additionally, the α2A adrenergic receptors, which are highly expressed in the LC, play a role in developing tau tangles through the GSK3β/tau signaling cascade (Braak and Del Tredici 2004; Ferrari et al. 2006; Khaliq and Bean 2010; Surmeier et al. 2017; Zhang et al. 2020; Matchett et al. 2021; Guzmán-Ramos et al. 2022). These molecular events and the characteristics of catecholaminergic neurons contribute to reduced LC innervation to other brain structures. This has been linked to decreased noradrenaline (NA) and DA levels in AD patients and mouse models, correlating with memory decline (Kelly et al. 2017). However, it is unclear whether restoring LC catecholaminergic transmission could alleviate the memory deficits associated with AD. To study the effects of stimulating LC catecholaminergic transmission on hippocampal synaptic plasticity and spatial memory retrieval, we selectively stimulated the catecholaminergic projections from the LC into the CA1 region of the dorsal hippocampus in a modified 3xTg-AD mouse model (AD-TH), which expresses the CRE recombinase protein under the control of the tyrosine hydroxylase (TH) promoter. We used optogenetic stimulation to activate the LC catecholaminergic projections into the CA1 hippocampal region before the retrieval session in two different spatial memory protocols: the Morris Water Maze (MWM) and Object Location Memory (OLM). Our findings indicate that optogenetic stimulation of the LC catecholaminergic projections into the CA1 hippocampal region before memory retrieval effectively reversed memory impairment in AD mice. Furthermore, we propose that the spatial memory retrieval deficit in AD-TH mice is partly due to decreased basal levels of catecholamines in the CA1 hippocampal region, which is associated with a reduced number of LC catecholaminergic projections and deficient synaptic plasticity in the same hippocampal region in aging. Interestingly, optogenetic stimulation of these LC catecholaminergic projections restored the levels of catecholamines and synaptic plasticity in the CA1 hippocampal region in these mice. These findings suggest potential therapeutic implications for targeting LC catecholaminergic pathways in AD-related cognitive decline. Materials and methods Subjects C57BL/6J wild-type mice (WT), along with 3xTgAD and TH-Cre x 3xTgAD (AD-TH) transgenic male and female mice, aged 3 (young) and 12 months (aging), were used during the optogenetic, electrophysiological, and behavioral experiments. The AD-TH strain was created by breeding 3xTgAD mice (Guzmán-Ramos et al. 2012; Moreno-Castilla et al. 2016) with TH-Cre mice (Gálvez-Márquez et al. 2022). Genotyping was performed to confirm the presence of the presenilin 1, amyloid precursor protein (APP), human tau, and Cre genes in the offspring. These mice were selectively bred for at least four generations alongside 3xTgAD mice to establish a strain expressing presenilin 1, APP, and tau proteins, with Cre-recombinase regulated by the TH promoter. For genotyping, we used the hotshot method. A tail snip was lysed in an alkaline reagent of 25 mM NaOH and 0.2 mM disodium EDTA at 95°C for 1 hour. After lysis, the DNA was neutralized using 1 M Tris-HCl (pH 7.4) and centrifugated at 2500 rpm for 2 minutes in a Hermie Z 233 MK-2 centrifuge. Finally, the DNA supernatant was collected. This supernatant was amplified using PCR (specific protocol number from QIAGEN) and analyzed via agarose gel electrophoresis. The mice were acquired from the Institute of Cellular Physiology animal facility at the National Autonomous University of Mexico. They were individually housed in transparent acrylic cages with ad libitum access to food and water, maintained under a 12/12-hour light/dark cycle at a temperature of 22 ± 2°C and relative humidity of 50 ± 5%. All experimental procedures were conducted during the light phase of the cycle, and ethical approval was obtained from the Institutional Animal Care and Use Committee of the Institute of Cellular Physiology (Approval No. FBR125-18), adhering to the guidelines outlined in the Official Mexican Standard (NOM-062-ZOO-1999). Stereotaxic surgery All animals were initially anesthetized using a mixture of oxygen (1L per min) and isoflurane (induction 5%; Maintenance 1–2%; Vip 3000 matrix) before being secured in a stereotaxic apparatus (RWM Life Science, Texas, USA). Surgical coordinates were referenced from the Allen Brain (Atlas Allen Institute for Brain Science). For optogenetic experiments, AD-TH mice were bilaterally injected with either Channelrhodopsin (ChR2) or empty viral vectors. Specifically, the ChR2 group received 0.5 µL per hemisphere of rAAV5/EfIα-DIO-hChR2 (viral concentration of 5.2 × 10 12 virus molecules/ml), while the control group received 0.5 µL per hemisphere of rAAV5/EfIα-DIO-eYFP (with a viral concentration of 6.0 × 10 12 virus molecules/ml, where eYFP stands for enhanced yellow fluorescent protein). The injections were targeted to the locus coeruleus (LC) at coordinates − 5.5 mm anterior-posterior (AP), ± 0.9 mm medial-lateral (ML), and − 3.3 mm dorsal-ventral (DV) relative to bregma. The virus was administered using calibrated glass micropipettes (5 µL, Drummond, USA). Additionally, optical fibers were bilaterally implanted in the dorsal hippocampus CA1 at coordinates − 2.40 mm AP, ± 2.0 mm ML, and − 1.00 mm DV relative to bregma. Mice used for the microdialysis experiments were implanted with an optical fiber (0.22 NA, 200 µm diameter; Doric Lenses, Canada) into the CA1 region of the hippocampus (− 2.40 mm AP; ±2.0 mm ML; −1.00 mm DV to bregma). A cannula guide (CMA/7; CMA Microdialysis, Sweden) was also bilaterally implanted into the CA1 (− 3.0 mm AP; ±2.0 mm ML; −1.5 mm DV to bregma) at a 25º frontal angle. After surgery, mice were allowed to recover for two weeks before beginning the experiments. Object Location Memory (OLM) task The OLM task was conducted in an open field inside a gray wooden box (33 x 33 x 30 cm) covered with a thin layer of sawdust. A spatial cue was positioned in one of the walls of the box. Two different Lego figures (5 x 5 x 5cm) were used as objects to be explored. Using Debut Video Capture-NCH software version 5.73, a video camera was positioned above the box to record the sessions. Mice were habituated to the box for 10 minutes without any objects for three consecutive days. During the following two acquisition sessions (10 minutes each day), the mice were allowed to explore two objects placed in specific positions. After a 24-hour interval, the long-term memory (LTM) test was conducted. During this test, the mice were allowed to explore the two objects for 10 minutes freely; however, one of the objects was relocated to a novel position for the trial. The novel and familiar positions of the objects were always counterbalanced to prevent any side preference in the arena. The boxes and objects were deodorized with 70% ethanol, and the sawdust bedding was changed between trials. The AD-TH mouse groups (eYFP and ChR2) received optogenetic stimulation (using a 473 nm laser at 10–15 watts and 20 Hz for 10 minutes) in the hippocampal CA1 region during the LTM test. The sessions were recorded and analyzed offline, focusing on the total exploration time for each object during the acquisition and LTM sessions. The recognition index was calculated by dividing the exploration time for each object by the total exploration time for both objects during each session. It was expected that the mice's recognition index would be close to 0.5 during the acquisition phase, indicating no preference for any object, in order to be included in the statistical analysis. Morris Water Maze (MWM) task A MWM behavioral task was conducted in a circular pool (110 cm in diameter) with a white bottom, where an escape platform (15 cm x 15 cm x 15 cm) was positioned at a fixed location 0.5 cm under the water level to remain invisible to the mice. Two geometric figures placed in opposite positions on the pool walls were used as spatial cues. A video camera was placed above the pool to record the trials during the acquisition and the LTM sessions using Debut Video Capture-NCH software version 5.73. Mice were handled for three minutes three days before the acquisition sessions to reduce stress. The acquisition sessions consisted of four training trials daily for four days. Animals were placed at different pre-established starting positions for each trial, with the experimenter always positioned in the same place, serving as a spatial cue. Mice were allowed to swim for 60 seconds and reach the escape platform. The time to reach the platform was recorded; if the mice could not find the escape platform within 60 seconds, the experimenter guided them. Once on the platform, the mice were allowed to explore for 30 seconds. After each trial, each mouse was placed in an open box for 60 seconds to rest before returning to the pool for the subsequent trial. The LTM test was conducted 48 hours after the last training session. During the LTM test, the escape platform was removed from the pool, and mice were allowed to swim freely for 60 seconds. AD-TH mouse groups (eYFP and ChR2) were optogenetically stimulated (473nm ser, 10 − 1,5 watts, 20 Hz, 5 minutes) in the hippocampal CA1 immediately before the LTM test (Kempadoo et al. 2016). Video records were analyzed offline to determine the number of platform crossings, time in the target quadrant, and swimming speed. The video was utilized to determine the area and location of the escape platform during the LTM sessions. The time each mouse spent reaching this area (latency to platform) and the number of times mice crossed to the area (number of crosses) were quantified. For the time analysis in the target quadrant, the pool was divided into four equal quadrants in the video, and the quadrant containing the escape platform was defined as the target quadrant. The swimming time of mice in the target quadrant was quantified. Swimming speed was quantified using the Image J tracking function. Electrophysiology The mice were anesthetized with sodium pentobarbital, initially at a dose of 20 mg/kg of body weight, and then a maintenance dose of 10 mg/kg was administered 60 minutes later. Once anesthetized, the mice were positioned in a stereotaxic apparatus (51603, Stoelting, Chicago, USA). A skin incision was made to expose the skull, and a stainless-steel concentric bipolar stimulation electrode was implanted in the CA3 region of the hippocampus (coordinates from bregma: AP 1.5 mm, ML 2.0 mm, DV 1.0 to 1.5 mm) using a unilateral trepan. A stainless-steel monopolar electrode was placed in the CA1 region of the hippocampus (coordinates from bregma: AP -2.2 mm, ML 1.4 mm, DV 1.0 to 1.5 mm) to record unilateral responses. Stimulation was provided using an AM Systems Model 2100 stimulator and delivered to the stimulation electrode. After establishing a baseline electrically evoked response for 15 minutes with a stimulation intensity set at 50% of the maximum EPSP amplitude, a long-term potentiation (LTP) protocol was initiated. This involved delivering four stimulation trains (1 second at 100 Hz) with 20-second intervals between each train, referred to as high-frequency stimulation (HFS). The evoked responses following this stimulation were recorded for an hour, and field excitatory post-synaptic potentials (fEPSP) were calculated as the percentage change in evoked response amplitude compared to the baseline. AD-TH mouse groups (eYFP and ChR2) were optogenetically stimulated using a 473 nm laser at 10–15 watts and 20 Hz for 5 minutes before the HFS and 10 minutes after the HFS. Offline analysis was conducted using the fEPSP recorded before and after the HFS. Microdialysis A CMA/7 membrane (CMA Microdialysis, Sweden) was inserted into the guide cannula previously implanted in mice. Ringer solution (MgCl2 12mM, NaCl 1.44 M, CaCl2 17 mM and KCl 48 mM) was perfused at a 0.25 µl/min rate using a micro-infusion continuous pump (100 pump CMA Microdialysis, Sweden). Mice were placed into the arena without objects and infused with Ringer solution to collect the neurotransmitter using the EICOM piping system (Concise Freely Moving System, EICOM, USA). Once the 30-minute stabilization period concluded, three 16-minute samples were collected to calculate the baseline. These samples were placed into a vial containing an antioxidant blend (Ascorbic acid 25 mM, Na2EDTA 27 mM, and acetic acid 1 M). Subsequently, a fourth fraction was collected while the mice (eYFP and ChR2) were optogenetically stimulated (473nm laser, 10–15 watts, 20 Hz, 16 minutes), and two post-stimulation samples were also collected. All samples were stored at -80ºC. Neurotransmitter analysis Neurotransmitter concentration was quantified by capillary electrophoresis. Briefly, all microdialysis samples were prepared for derivatization by adding 6µL of 3-(2- complete)-quinoline-2-carboxaldehyde (FQ, 16.67 mM, Molecular Probes; Massachusetts Invitrogen, USA) and catalyzed with 2µL KCN (24.5 mM) in borate buffer (10 mM pH 9.2). 1 µL of an internal standard (0.075 mM, O-methyl-L-threonine; Fluka, Indiana, USA) was added to each microdialysis sample, and they were incubated in the dark for 15 min at 65ºC. Neurotransmitters were quantified with laser-induced within a capillary electrophoresis system (P/ACE MDQ, Beckman Coulter; Pasadena, USA). Compound separation was based on the micelle electrokinetic chromatography method. Microdialysis samples were hydro-dynamically injected into the capillary system at 0.5 psi for 5s. Separation occurred in a buffer (borates 35 mM, sodium dodecyl sulfate 25 mM, and 13% methanol HPLC grade, pH 9.6) at neurotransmitters was detected by fluorescence using a LIF device (488nm). Signals were depicted as electropherograms and analyzed offline using 32Karat TM8.0 software (Beckman Coulter, Pasadena, USA). Neurotransmitters were identified by comparison with the standard electropherogram pattern (DA and NA standards). Signals were quantified by measuring the ratio of the area under the curve for each neurotransmitter and the area under the curve of its respective internal standard. Immunofluorescence and confocal microscopy Mice were euthanized utilizing sodium pentobarbital (75 mg/kg) after the behavioral, electrophysiological, and neurotransmitter analysis experiments. Immediately after euthanasia, mice were perfused with a 0.9% saline solution and fixed with a 4% paraformaldehyde solution. The brain was extracted and preserved in a 4% paraformaldehyde solution. A cryostat (Leica CM520, Germany) cut mice brains in 40 µm coronal slices. Free-floating tissue slices were incubated overnight with primary antibodies diluted in a 5% bovine serum albumin buffer (NaCl 150 mM, Triton X-100 0.1%, Trizma base 100 mM, pH 7.4) at a dilution of 1:1000 (mouse monoclonal anti-human phospho-PHF-tau pSer202/Thr205 antibody, Thermo Scientific, Belgium; mouse monoclonal BAM-10 anti-beta-amyloid antibody, Missouri, Sigma Aldrich, USA; rabbit polyclonal anti-TH antibody, Arkansas, Pel-Freez, USA). In the case of immunohistochemistry against Aβ, the floating tissues were incubated for 5 minutes in formic acid and washed with TBS-T 0.1% before the primary antibody incubation. The next day, floating slices were washed 6 times with Tris-buffered saline solution and then incubated with secondary antibodies diluted in 5% bovine serum albumin buffer at a 1:500 dilution (Mouse IgG antibody (FITC), California, GeneTex, USA; Gt X Rb IgG Cy3, Massachusetts, Millipore, USA) for 2 hours. After that, the slices were mounted using a Dako fluorescence mounting medium. The immunofluorescence was examined using a ZEISS LSM 800 confocal microscope (Zeiss, Germany). The immunofluorescence images were digitized for further analysis using Image J (version), and the antibody signal was obtained and quantified by measuring the percentage of the pixel area. Statistics Statistics analysis was conducted using GraphPad Prism software (version 7.00, USA). All graphs showed mean ± sem with a statistical significance of p < 0.05. For immunohistochemistry, images were processed with Image J software and analyzed with One-way ANOVA and Tukey post hoc. OLM and MWM data were analyzed with one or two-factor ANOVA, followed by multiple comparison tests with statistical significance determined using the Fisher's LSD or Tukey tests. Microdialysis analyses were conducted using one-way ANOVA and Holm-Sidak's post hoc analysis. One-way ANOVA and Holm-Sidak's post hoc analysis were performed for electrophysiology. Results Optogenetic stimulation of the LC-CA1 projections enhances spatial memory retrieval in an AD-TH mouse model of Alzheimer's disease Research has demonstrated the importance of catecholamines in spatial memory, mainly through optogenetic techniques. These studies have shown that inhibiting the catecholaminergic projections from the LC to the hippocampal CA1 region leads to impaired spatial memory expression (Gálvez-Márquez et al. 2022). Notably, a reduction in these projections has been observed in the early stages of AD (Theofilas et al. 2017; James et al. 2021), which motivated us to investigate further the effects of optogenetic activation of the LC projections on hippocampal-dependent memory and synaptic plasticity in a murine model of AD. To achieve this, we used an adenoviral vector to express the ChR2 protein or the enhanced eYFP reporter protein in LC neurons. Additionally, we implanted an optic fiber in the CA1 region of the hippocampus in our modified AD-TH mouse model (Fig. 1 a and b). To evaluate the viral infection efficacy in the AD-TH mice's LC neurons and their hippocampal projections (Fig. 1 a and b), we conducted an immunohistochemical analysis of the eYFP reporter protein. This analysis assessed its expression in LC TH + neurons and their projections within the CA1 hippocampal region. As shown in Fig. 1 c, the eYFP reporter protein is expressed in the LC neurons that project to the CA1 hippocampal region. Furthermore, the TH immunoreactive signal colocalizes with the eYFP immunoreactive signal in both brain regions (Fig. 1 c). We implemented a behavioral paradigm to assess hippocampal-dependent OLM (Fig. 1 d) and to evaluate the effect of the optogenetic stimulation of the catecholaminergic LC projections within the CA1. During the two-day acquisition sessions, all groups of mice exhibited an equal exploration of the two objects, evidenced by their recognition index, demonstrating no preference to explore either object (Fig. 1 e). Twenty-four hours post-acquisition, the AD-TH mice underwent optogenetic stimulation during a 10-minute LTM session. While WT mice recognized the object in a novel location compared to a familiar one, both 3xTgAD and AD-TH eYFP mice failed to show recognition of the object in the novel location versus the familiar location. This indicates a deficit in retrieving LTM in OLM, likely due to the AD pathology. In contrast, optogenetic stimulation of the catecholaminergic LC projections within CA1 improved the retrieval of OLM in AD-TH mice expressing ChR2, demonstrated by a higher recognition index for the object in the novel location than the familiar location (Fig. 1 f). Additionally, young AD mice and young AD-TH could retrieve LTM in OLM, evidenced by a preference to explore the object in a novel location vs a familiar location (Supplementary Fig. 1a, b, and c). At the same time, no difference in the total exploration time was observed (Supplementary Fig. 1d). A MWM behavioral protocol was utilized to corroborate the effect of optogenetic activation of the catecholaminergic LC-CA1 projection on the recovery of spatial memory retrieval. Both young and aging mice were included in the behavioral tests. Four training trials were conducted daily over four days during the acquisition phase. Throughout these sessions, no differences were observed in the latency time to reach the hidden platform among young and old WT, 3xTgAD, and AD-TH mice across the training days. By the fourth day of training, all groups of mice showed reduced latency times to reach the hidden platform (Fig. 2 b, and Supplementary Fig. 2b). Forty-eight hours later, during the LTM test, young WT, AD-TH, and 3xTgAD groups showed strong spatial memory retrieval (Fig. 2 c, and Supplementary Fig. 2c). However, aging AD mice exhibited deficits in spatial memory retrieval compared to their same-aged WT counterparts. The 3xTgAD and AD-TH eYFP mice showed a reduced number of crossings to the platform area (Fig. 2 c), a lower percentage of time spent in the target quadrant (Fig. 2 d), and longer latency times to reach the platform area (Fig. 2 e). These findings indicate a compromised ability to retrieve spatial memory in these groups. Interestingly, the AD-TH ChR2 mice exhibited enhanced spatial memory retrieval when optogenetic stimulation of the catecholaminergic projections from the locus coeruleus to the CA1 region was applied just before testing for long-term memory (LTM). The results showed a greater number of area crossings, indicating a recovery effect. These mice also spent a larger percentage of time in the target quadrant and displayed a shorter latency to the platform area compared to the AD-TH eYFP mice, achieving results like those of the WT mice (see Fig. 2 c, d, and e). Importantly, swimming speed remained consistent across all groups, with no changes resulting from the optogenetic stimulation of the catecholaminergic LC projections in the CA1 hippocampal region (Fig. 2 f). These findings suggest that while memory acquisition is unaffected in young and aging AD mice, memory retrieval performance in the MWM and OLM tasks is impaired in aging AD mice. However, activating the catecholaminergic LC projections within the CA1 region of the hippocampus enhances spatial memory retrieval, as evaluated through the OLM and MWM paradigms. This evidence supports the notion that deficiencies in spatial memory retrieval are age-dependent, but activating the catecholaminergic LC projections in the CA1 region is sufficient to mitigate these retrieval deficits in older AD mice. Optogenetic stimulation of the LC-CA1 projections enables LTP induction in AD mice Reports have shown alterations in hippocampal synaptic plasticity in AD mice (Oddo et al. 2003; Tönnies and Trushina 2017; Brandwein and Nguyen 2019). To assess whether the enhancement of the catecholaminergic LC neurotransmission within the hippocampus improves synaptic plasticity, mice were injected with the adenoviral vector carrying ChR2 or eYFP reporter proteins in the LC (Fig. 3 a). Young and aging AD-TH mice were subjected to LTP protocol coupled with the optogenetic stimulation of the catecholaminergic LC projections in the Shaffer collateral pathway of AD mice under anesthesia (Fig. 3 b). LTP was induced by applying an HFS (3 trains of 100 pulses at 100Hz) (Fig. 3 c) in the CA3 projections. At the same time, the field fEPSP was measured within CA1 of the hippocampus. Interestingly, young AD mice could induce and maintain a strong LTP (Supplementary Fig. 3c and e), suggesting that alterations in the hippocampal synaptic plasticity in AD are aging dependent. In aging mice, WT mice exhibited an augmented fEPSP after HFS compared to the baseline recordings (137.5% ± 12.07%), and these responses persisted for up to one hour (Fig. 3 d). In contrast, 3xTgAD mice failed to induce LTP; instead, a depression in the fEPSP recordings following HFS was observed (85.41% ± 9.05%) (Fig. 3 e). Similarly, eYFP AD-TH mice presented a decrement in the synaptic communication post-HFS (78.01.7% ± 10.35%) (Fig. 3 f). However, the optogenetic stimulation of the catecholaminergic LC projections within the CA1 hippocampal region of ChR2 AD-TH mice reinstated the synaptic plasticity within the Schaffer collaterals pathway (152.7% ± 17.47%) after HFS (Fig. 3 g). This observed potentiation of the fEPSP in ChR2 AD-TH mice was similar to that in WT mice (Fig. 3 h). These results indicate that enhancing the catecholaminergic LC neurotransmission within the CA1 hippocampus significantly improves the synaptic plasticity in that specific brain region. Interestingly, this reinstatement of the synaptic plasticity in the hippocampus is associated with the improved ability of AD mice to retrieve spatial memory effectively. Optogenetic stimulation of the LC-CA1 projections increases dopamine and noradrenaline levels in AD-TH mice Research has shown that catecholaminergic neurotransmitters in the hippocampus are reduced in AD mouse models and patients (Kelly et al., 2017; Theofilas et al., 2017; Dahl et al., 2023). This raises an important question: does optogenetic stimulation of the catecholaminergic projections from the locus coeruleus to the CA1 region increase DA and NA levels in an AD mouse model? To investigate this possibility, we conducted capillary electrophoresis analysis using an in vivo microdialysis approach to quantify catecholaminergic neurotransmitter levels. Adenoviral vectors encoding the Channelrhodopsin-2 (ChR2) protein or the enhanced yellow fluorescent protein (eYFP) reporter protein were injected into the locus coeruleus of AD-TH mice. Simultaneously, neurotransmitter samples were collected from the CA1 region of the hippocampus during the optogenetic stimulation process (Fig. 4 a and b). Neurotransmitter samples were obtained at three different time points: before the stimulation (BASAL), during the stimulation (STIM), and after the stimulation at two intervals (POST1 and POST2) (Fig. 4 c). The quantification of NA and DA levels showed a significant reduction in the CA1 hippocampus of 3xTgAD and AD-TH mice compared to WT mice. The optogenetic stimulation of the LC projections to the CA1 hippocampus successfully restored extracellular levels of NA and DA for up to 30 minutes following the stimulation (Fig. 4 d and e). These findings indicate that DA and NA concentrations are diminished in the hippocampus of AD mice. This reduction and other pathological processes associated with AD contribute to deficits in spatial memory retrieval. Notably, restoring catecholaminergic neurotransmission from the LC to the hippocampus is sufficient to enhance catecholaminergic levels and improve spatial memory retrieval in AD mice. Phenotypical characterization of the AD-TH mice The AD-TH mouse model was developed to optogenetically activate the catecholaminergic LC projections into the CA1 region of the hippocampus by interbreeding 3xTgAD mice with TH-Cre mice. First, genotyping confirms the presence of presenilin 1, APP, human tau, and Cre genes in AD-TH mice. Then, it was necessary to describe the phenotypic expression of hallmark proteins associated with AD, specifically the Aβ protein and hyperphosphorylated tau protein. These proteins are known to be overexpressed and accumulate within both the LC and the hippocampus in the context of AD pathology. In our analysis, we also examined TH protein levels in both brain structures to quantify the catecholaminergic neuron somas in the LC and the catecholaminergic terminals in the hippocampus of aging mice. A significant increase in Aβ protein (Fig. 5 a and d) and hyperphosphorylated tau protein (Fig. 5 b and e) was observed in the 3xTgAD and AD-TH mice within the CA1 hippocampus compared to WT mice. However, no significant differences in the expression of these proteins were found between the two AD mouse models. Additionally, the TH immunoreactive signal in the hippocampus indicated reduced catecholaminergic projections (Fig. 5 c and f). Similarly, Aβ (Fig. 5 g and j) and hyperphosphorylated tau (Fig. 4 h and k) proteins showed increased immunoreactive signals in the LC of aging 3xTgAD and AD-TH mouse models. While both proteins were overexpressed in the LC, no significant difference in the immunoreactive signal for TH was found in the neuron somas of WT, 3xTgAD, and AD-TH mice (Fig. 5 i and l). A comparative analysis of the Aβ protein immunoreactive signals between the CA1 hippocampus region and LC indicates a more significant accumulation of Aβ proteins in the hippocampus compared to the LC (LC vs. HIP: t( 6 ) = 8.992, p = 0.0001). In contrast, the immunoreactivity of hyperphosphorylated tau protein was significantly higher in the LC than in the hippocampus (LC vs. HIP: t( 6 ) = 3.469, p = 0.0133). These findings suggest that the expression and accumulation of Aβ and hyperphosphorylated tau proteins occur differentially. While TH-AD mice exhibit detectable accumulation of both proteins, Aβ primarily accumulates in the CA1 region of the hippocampus. Conversely, hyperphosphorylated tau protein mainly accumulates in the LC of old AD mice. Furthermore, our phenotypic analysis of catecholaminergic neurons in the LC and their projections to the hippocampus implies that the reduction of catecholaminergic neurotransmitter levels in the hippocampus is due to a decline in the catecholaminergic projections rather than a loss of catecholaminergic neurons in the LC. Discussion Alzheimer's disease progression involves ongoing damage to neuronal function in both the cortical and hippocampus regions. Notably, mice models of Alzheimer's display significant degeneration of catecholaminergic terminals in the hippocampus around 12 months of age. This damage is followed by neuronal degeneration in the LC and the VTA at 18 and 24 months, respectively. This progressive degeneration of catecholaminergic activity is primarily attributed to the deposition of Aβ peptide (Grudzien et al. 2007; Liu et al. 2008; Moreno-Castilla et al. 2016). They also underscore its significance in developing cognitive deficits associated with AD (Braak and Del Tredici 2016; Hansen 2017; Dahl et al. 2023). Recent research suggests a denser catecholaminergic projection from LC into the CA1 of the hippocampus, as opposed to sparse dopaminergic fibers originating from the VTA (Takeuchi et al. 2016; Kempadoo et al. 2016; Tsetsenis et al. 2022; Gálvez-Márquez et al. 2022). It has been reported that projections from the LC are mainly located in the dorsal regions of the hippocampus, which are relevant for memory processes. In contrast, dopaminergic innervation from the VTA targets the ventral areas of the hippocampus (Titulaer et al. 2021; Wilmot et al. 2024). The LC catecholaminergic projections to the hippocampus have been recognized as pivotal in modulating synaptic plasticity and cellular mechanisms underlying memory (Lemon and Manahan-Vaughan 2012; Mravec et al. 2014; Hansen and Manahan-Vaughan 2015; Gálvez-Márquez et al. 2022). Particularly in the CA1 region, this catecholaminergic role is crucial for restoring place cells accountable for spatial details within the hippocampus (Kaufman et al. 2020). These findings emphasize the significance of LC-hippocampus catecholaminergic neuromodulation in regulating hippocampal-dependent memory processes. Therefore, this abnormally low level of LC catecholaminergic projections into the CA1 region provides a pathological substrate for the retrieval deficiencies observed in AD mice. This reduction in LC catecholaminergic projections within the same hippocampal region has been observed in AD patients and murine models (Reinikainen et al. 1988; Chen et al. 2022). Optogenetic activation of the LC-CA1 projections improves the catecholaminergic neurotransmission within the hippocampus of AD-TH mice In our research, we observed a significant decrease in the baseline levels of DA and NA in the CA1 area of the hippocampus in 3xTg-AD and AD-TH mice. The reduced levels of these neurotransmitters in the hippocampus of AD patients are thought to be part of the cognitive dysregulation that causes AD symptoms(Lyness et al. 2003; Francis et al. 2012; Hagena et al. 2016). Our findings show that the decrease in DA and NA levels in the CA1 region is linked to a reduction in hippocampal CA1 catecholaminergic projections from the LC rather than a loss of LC catecholaminergic neurons, at least in 12-month-old mice. We also showed that optogenetic stimulation of the CA1 hippocampal catecholaminergic projections from the LC is enough to restore hippocampal catecholaminergic levels. The LC is a crucial brain region where abnormal hyperphosphorylated tau protein accumulates in individuals with AD (Mather and Harley 2016; Beardmore et al. 2021). In both of our transgenic mouse models, the levels of hyperphosphorylated tau protein in the LC align with Braak staging and have been detected before the onset of tau pathology in the hippocampus (Grudzien et al. 2007; Braak and Del Tredici 2016). Although we did not observe a decrease in TH + neurons in the LC of our 12-month-old AD mice—an indication typically associated with mid- to late-stage AD—the accumulation of abnormal tau protein could still lead to degeneration of catecholaminergic projections. This degeneration may result in reduced basal release of DA and NA in the hippocampus, which are necessary to retrieve OLM (Moreno-Castilla et al. 2016; Gálvez-Márquez et al. 2022). Additionally, the Aβ peptide plays a neuromodulatory role in regulating synaptic communication and neurotransmitter release (Karisetty et al. 2020), mediated through direct and indirect interactions with presynaptic proteins that control neurotransmitter vesicle release (Abramov et al. 2009; Russell et al. 2012; Gulisano et al. 2019). However, the accumulation of Aβ peptide has a detrimental effect on neurotransmitter release. The combination of diminished LC catecholaminergic projections and the potential adverse consequences of Aβ peptide accumulation may contribute to the diminished catecholaminergic levels observed here, thereby impeding the hippocampal synaptic efficiency and negatively impacting the retrieval of spatial memories in AD mice. Nevertheless, we show that the pathological low level of catecholamines in the hippocampus can be reversed through the optogenetic stimulation of the LC catecholaminergic projections into the CA1 hippocampal region in the AD-TH mice. Optogenetic stimulation of the LC-CA1 projections reverses spatial memory retrieval deficiencies in AD mice Research on Alzheimer's Disease suggests that the primary challenge lies in retrieving memories rather than storing them (Roy et al. 2016; Jura et al. 2019; Small and Cochrane 2020; Bostancıklıoğlu 2020). Our findings support this hypothesis. We observed that young AD mice can remember and recall spatial information in both tasks. However, aging mice from the 3xTg-AD and AD-TH models struggle to retrieve this information during LTM assessments. We found that stimulating the catecholaminergic projections from the LC-CA1 of the hippocampus before LTM tasks significantly improved memory retrieval in our AD-TH mouse model, and additionally, activating these catecholaminergic projections in the same CA1 region before retrieval enhanced the ability of the mice to recognize new object locations during LTM sessions. These results suggest that AD-TH mice can form and consolidate hippocampal-dependent memories but face challenges in retrieving these memories. Accordingly, we demonstrated that photoinhibition of the LC catecholaminergic projections into the CA1 hippocampus prevents the retrieval of the spatial memory (Gálvez-Márquez et al. 2022), highlighting the importance of the catecholaminergic activity from the LC to retrieve the spatial memory in the hippocampus adequately. Administering a DA D1-like receptor antagonist or a beta-adrenergic receptor antagonist before a LTM session in the CA1 region of the hippocampus impairs spatial memory retrieval. However, DA activity—rather than NA activity—is crucial for updating spatial memory (Gálvez-Márquez et al. 2022). In this context, administering NA precursors or DA agonists has been shown to reduce the toxic effects of Aβ and improve performance in spatial memory tasks (Himeno et al. 2011; Kalinin et al. 2012; Gutiérrez et al. 2022). Research also indicates that blocking DA reuptake in the cortex of AD transgenic mice can alleviate memory recognition impairment and enhance dopaminergic activity (Guzmán-Ramos et al. 2012, 2022; Moreno-Castilla et al. 2016). Together, these studies emphasize that catecholaminergic activity is closely related to AD dysfunction, representing a potential neurochemical target for pharmacological treatment. Consequently, using DA agonists, rotigotine, the DA precursor L-DOPA or the monoamine oxidase-B inhibitor selegiline has enhanced synaptic plasticity and improved memory deficits in experimental AD models and humans (Koch et al. 2014; Dahl et al. 2023). Although further investigation is needed to identify the specific catecholaminergic receptors involved in memory retrieval, these findings suggest that stimulating catecholaminergic pathways in the brain could help restore memory retrieval in 12-month-old AD-TH mice. Additionally, these results align with other studies indicating that Alzheimer's patients often struggle with memory recall due to difficulties in activating memory patterns. Notably, this study is the first to highlight the critical and sufficient role of LC catecholaminergic activity in the hippocampus for enhancing memory retrieval in models of Alzheimer's disease. Optogenetic activation of the LC-CA1 projections restores synaptic plasticity in AD-TH mice. Our experimental approach indicates that enhancing spatial memory retrieval in early-stage AD mice may be linked to promoting hippocampal neural plasticity. This improvement could result from restoring catecholaminergic levels through optogenetic stimulation of the LC catecholaminergic projections within the CA1 region of the hippocampus. It is well known that DA and NA's transient bursts (phasic activity) occur in response to rewarding or novel stimuli to promote LTM (Harley 2004). Accordingly, younger AD mice demonstrate normal synaptic plasticity, but by age, they can no longer induce LTP following high-frequency stimulation. These findings suggest that the age of the AD mice influences the inability to enable synaptic plasticity. Catecholamines play a crucial role in modulating the molecular and cellular mechanisms that contribute to synaptic plasticity through their specific receptors (Twarkowski and Manahan-Vaughan 2016; Madadi Asl et al. 2019). A reduction in innervation can lead to disinhibition and increased excitability in hippocampal areas, often observed in the early stages of Alzheimer's disease (Goettemoeller et al. 2024). NA interacts with α1, α2, and β adrenergic G-protein coupled receptors (GPCRs), activating Gs subunits and cAMP pathways significantly influencing synaptic plasticity (Marzo et al. 2009; Maity et al. 2020, 2022). Additionally, NA can induce hyperpolarization in cortical neurons (Wong et al. 2023) and enhance LTP in the CA1 region by promoting protein kinase A (PKA) pathways (Gelinas et al. 2008) and activating guanine nucleotide exchange proteins through cyclic adenosine monophosphate (cAMP)(Brandwein and Nguyen 2019). DA influences synaptic function through D1-like and D2-like G protein-coupled receptors GPCRs, which promote LTP and LTD in the hippocampus, respectively (Caragea and Manahan-Vaughan 2021; Kim et al. 2022). While ionotropic receptors mediate rapid neuronal responses through the immediate flow of ions across the cell membrane, GPCRs trigger a slower activation of intracellular signaling cascades. This process indirectly affects ion and neurotransmitter release, ultimately influencing gene expression and synaptic plasticity (Betke et al. 2012). Although hippocampal CA1 synaptic plasticity in the AD-TH mice was reinstated due to optogenetic stimulation of the LC catecholaminergic projections, we observed a slow onset in the induction of LTP. As mentioned earlier, this slow onset may be attributed to the delayed response elicited by the GPCRs (Greengard 2001; Wong et al. 2023; Tse et al. 2023). Recent studies indicate that synaptic plasticity in the hippocampus can be influenced by optogenetic stimulation of catecholaminergic projections in healthy mice (Takeuchi et al. 2016; Kempadoo et al. 2016; Gálvez-Márquez et al. 2022). The activity of catecholamines from the LC highlights the importance of neural communication within the hippocampus (Hansen 2017). Recent findings demonstrate that photoinhibition of hippocampal catecholamine projections from the LC can alter the threshold for transitioning from LTP to LTD following high-frequency stimulation (Gálvez-Márquez et al. 2022). This change may be linked to decreased levels of DA and NA. Additionally, DA depletion caused by Aβ peptide has been shown to impair synaptic plasticity, converting high-frequency stimulation-induced LTP into LTD in the dorsal hippocampus (Mayordomo-Cava et al. 2020) and cortical circuits (Moreno-Castilla et al. 2016). Similarly, the co-administration of DA and beta-adrenergic receptor antagonists can induce LTD following high-frequency stimulation (Gálvez-Márquez et al. 2022). Interestingly, we have found that increased cortical DA activity can reverse LTD induced by Aβ1–42 oligomers back into LTP (Guzmán-Ramos et al. 2012, 2022; Moreno-Castilla et al. 2016). These results align with the observed rise in catecholamines when photostimulation of the LC-CA1 terminals leads to improved memory and a shift from LTD to LTP in the hippocampal CA1 region. Our experimental approach suggests stimulating the catecholaminergic system is vital for forming LTM by enhancing synaptic plasticity mechanisms. These changes are essential for activating and triggering the memory engram, enabling AD-TH mice to retrieve previously acquired spatial information. Conclusion The exact causes and underlying mechanisms of Alzheimer’s disease are not yet fully understood. However, it is essential to develop strategies to improve the memory deficits associated with AD for clinical treatments. Some experimental approaches suggest that stimulating the catecholaminergic system through pharmacological methods (Koch et al. 2014; Shaikh et al. 2023) or repeated exposure to novel stimuli could be critical for cognitive improvement (Velázquez-Delgado et al. 2024). These strategies emphasize the influence of lifestyle and environmental factors on AD and their potential to alleviate symptoms. Research indicates that abnormal tau aggregation in the LC and dysfunction of the catecholaminergic system may represent the early stages of AD progression. Our findings suggest that therapies targeting the projections from the LC to the cortex and hippocampus could enhance cognitive function in patients with mild cognitive impairment due to early AD. These results highlight the significance of targeting LC catecholaminergic neurons as potential therapeutic targets to address cognitive deficits in AD patients through targeted stimulation. Abbreviations AD, Alzheimer´s disease.AD-TH, Alzheimer´s disease-Tyrosine hydroxylase . APP, Amyloid precursor protein.Aβ, Beta-amyloid peptide. ChR2, Channelrhodopsin. eYFP, DA, Dopamine . Enhanced yellow fluorescent protein.EPSP, Excitatory post-synaptic potentials. LC, Locus Coeruleus. LTM, Long-Term memory.LTP, Long-Term potentiation. MWM, Morris Water Maze. NA, Noradrenaline.OLM, Object Location Memory . TH, Tyrosine hydroxylase . 3Tg-AD, Triple transgenic Alzheimer mouse model. VTA, Ventral Tegmental Area. WT, Wild type. Declarations Acknowledgments and Funding . We would like to express our gratitude to the Unidad de Imagenología at the Instituto de Fisiología Celular, UNAM, particularly to Dr. Ruth Rincón Heredia and Dr. Abraham Rosas Arellano, for their invaluable technical support. We also thank Psychologist Ana Cecilia López Sepúlveda for her assistance in conducting the behavioral and electrophysiological experiments. This study was performed as part of the requirements requested to obtain a doctoral degree in Biochemical Sciences for Donovan K. Gálvez-Márquez at UNAM. D.K.G.-M. received the fellowship 856256 from Consejo Nacional de Humanidades Ciencia y Tecnología (CONAHCYT), México. This project was supported by the CONAHCyT grants FOINS 474, CF-2023-I-189, and DGAPA-PAPIIT-UNAM grant IN 213123 to F.B.-R. Author contributions . D.K.G.-M.,O.U.-M., L.R.-D., and F.B.-R. designed research; D.K.G.-M. and O.U.-M. performed research; D.K.G.-M., O.U.-M., L.R.-D., and F.B.-R. analyzed data; and D.K.G.-M.,O.U.-M. and F.B.-R. wrote the paper. Conflict of interest disclosure. The authors declare no conflicts of interest. Data Availability . Analyzed data will be made available upon reasonable request to the corresponding author. Ethical Approval . 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Circuito Exterior, Ciudad Universitaria, 04510, Mexico City, Mexico.","correspondingAuthor":false,"prefix":"","firstName":"Donovan","middleName":"K.","lastName":"Gálvez-Márquez","suffix":""},{"id":404739857,"identity":"8ec9b5b9-6398-4ca9-9e5e-47e48796dc60","order_by":1,"name":"Oscar Urrego-Morales","email":"","orcid":"","institution":"División de Neurociencias. Instituto de Fisiología Celular. Universidad Nacional Autónoma de México. Circuito Exterior, Ciudad Universitaria, 04510, Mexico City, Mexico.","correspondingAuthor":false,"prefix":"","firstName":"Oscar","middleName":"","lastName":"Urrego-Morales","suffix":""},{"id":404739858,"identity":"ef1b0f81-eb6f-4d01-b0b4-cd73c86d157b","order_by":2,"name":"Luis F. Rodríguez-Durán","email":"","orcid":"","institution":"División de Neurociencias. Instituto de Fisiología Celular. Universidad Nacional Autónoma de México. Circuito Exterior, Ciudad Universitaria, 04510, Mexico City, Mexico.","correspondingAuthor":false,"prefix":"","firstName":"Luis","middleName":"F.","lastName":"Rodríguez-Durán","suffix":""},{"id":404739859,"identity":"778ac370-0c6d-40d4-969f-2aba58cf9a24","order_by":3,"name":"Federico Bermúdez-Rattoni","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA1ElEQVRIiWNgGAWjYBAC9hlgipmBn5nxAXFaeG5AtUg2MxuQqMXgANFapJuPfbrZZi1nfJyZ8QNj2x153QbuNAm8WmSOJc/ObUs3NjvMzCzB2PbMcNsB3s147bOXyDFmzm07nLjtMP8xBsa2w4xALRsf4LVFIv8zSEv95mZmNpAWe6CWDQfwa8lhBmlJMGCGaEkkbIvMMWPmnHPphjNAfkk4dzh522ECfgGG2GPmnDJref7+w4wfPpQdtt12vHcb3hBDBQkggpl49aNgFIyCUTAKcAAAbvZFcpvIHnQAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0003-2056-6119","institution":"División de Neurociencias. Instituto de Fisiología Celular. Universidad Nacional Autónoma de México. Circuito Exterior, Ciudad Universitaria, 04510, Mexico City, Mexico.","correspondingAuthor":true,"prefix":"","firstName":"Federico","middleName":"","lastName":"Bermúdez-Rattoni","suffix":""}],"badges":[],"createdAt":"2025-01-20 19:03:35","currentVersionCode":1,"declarations":{"humanSubjects":false,"vertebrateSubjects":false,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":false},"doi":"10.21203/rs.3.rs-5868268/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5868268/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":74449792,"identity":"8a17c701-d804-49fc-a00a-b60d3ea426ed","added_by":"auto","created_at":"2025-01-22 11:41:24","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":15444714,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOptogenetic stimulation of catecholaminergic LC-CA1 projection improves memory retrieval in AD-TH mice in OLM protocol.\u003c/strong\u003e \u003cstrong\u003ea\u003c/strong\u003e Schematic representation of the bilateral adenoviral injection of AAV-ChR2 or AAV-eYFP within the LC in AD-TH mice. \u003cstrong\u003eb \u003c/strong\u003eThe viral construct of AAV-ChR2 and a diagram of the CRE recombinase protein under the control of the TH promoter are depicted. \u003cstrong\u003ec \u003c/strong\u003eRepresentative images of coronal sections of LC and hippocampal CA1 projection from LC are shown. eYFP (green), TH (red), DAPI (blue), and MERGE (colocalization). \u003cstrong\u003ed\u003c/strong\u003e Schematic diagram of the OLM protocol. Optogenetic stimulation of the catecholaminergic LC projections within the CA1 hippocampal region was delivered along the 10-minute LTM session (20 Hz, 5 ms, 473 nm to 10-15 mW). \u003cstrong\u003ee\u003c/strong\u003e Average of the recognition index during the acquisition sessions for the WT (n=11), 3xTgAD (n=11), and AD-TH eYFP (n=4) AD-TH ChR2 (n=5) mice groups. No preference to explore either of the objects is observed in any of the mice groups (Acquisition; Two-way- ANOVA: groups factor, F (3,58) \u0026lt; 0.0001, P \u0026gt; 0.9999; between objects, F(1,58) = 0.9169, P = 0.3423 ). \u003cstrong\u003ef \u003c/strong\u003eDuring the LTM, the location of one object changed (NL) (LTM; Two-way- ANOVA: groups factor, F (3,56) \u0026lt; 0.0001, P \u0026gt; 0.9999; between objects, F (1,56) = 19.65, P \u0026lt; 0.0001). WT mice (average exploration time: 790 ± 102 cs) recognized the object in novel location vs. familiar location (Fisher’s LSD, t(56) = 2.243, P = 0.0289), however 3xTgAD (average exploration time: 562 ± 116 cs) and AD-TH eYFP mice (average exploration time: 717 ± 163 cs) failed to recognized the object in novel location vs familiar location (Fisher’s LSD, 3xTgAD: t(56) = 1.735, P = 0.0883, AD-TH eYFP: t(56) = 0.3577, P = 0.7219). Finally, AD-TH ChR2 mice (average exploration time: 278 ± 79 cs) recognized the object in a novel location vs. a familiar location (Fisher’s LSD, t (56) = 7.038, P \u0026lt; 0.0001). The red dotted horizontal line represents the 0.5 recognition index threshold. The blue bars indicate the optogenetic stimulation. All results showed the mean ± SEM. *P \u0026lt; 0.05. HPC: Hippocampus; cs: centi-seconds. Scale bar: 50 µm.\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-5868268/v1/0a40f437c35caecce0edb02f.png"},{"id":74449790,"identity":"b721b8e9-4890-4f87-b4ed-9b7986f657a2","added_by":"auto","created_at":"2025-01-22 11:41:24","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":5277089,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOptogenetic stimulation of catecholaminergic LC-CA1 projection reverses the retrieval deficits in the MWM spatial memory in AD-TH mice.\u003c/strong\u003e \u003cstrong\u003ea\u003c/strong\u003e Schematic diagram of the MWM protocol. Optogenetic stimulation was delivered 5 minutes before the LTM test for the \u0026nbsp;AD-TH ChR2 and AD-TH eYFP mice groups. \u003cstrong\u003eb\u003c/strong\u003e Latency time to the hidden platform during the four days acquisition session for WT (n = 10), 3xTgAD (n = 11), AD-TH ChR2 (n = 9) and AD-TH eYFP (n = 6) mice are shown. All mice groups displayed improved performance to reach the hidden platform over time, and no statistical differences were observed between mice groups (Repat measures two-way ANOVA, groups F (3,31) = 0.4566, P = 0.7146; time F(3,93) \u0026nbsp;= \u0026nbsp;55.79, P \u0026lt; 0.0001) (Fishers LSD, WT latency time acquisition 1 day vs. 4 days: t(93) = 6.377, P \u0026lt; 0.0001; 3xTgAD latency time acquisition 1 day vs. 4 days: t(93) = 5.239, P \u0026lt; 0.0001; AD-TH eYFP latency time acquisition 1 day vs. 4 days: t(93) = 5.085, P \u0026lt; 0.0001; AD-TH ChR2 latency time acquisition 1 day vs. 4 days: t(93) = 7.774, P \u0026lt; 0.0001). Forty-eight hours later, a LTM session was performed, and the following parameters were measured. \u003cstrong\u003ec\u003c/strong\u003e Number of crosses to the platform area (One-way ANOVA, F (3,33) = 8.945, P = 0.0002). The 3xTgAD and AD-TH eYFP mice exhibited a significantly lower number of crosses to the platform area than WT mice. However, AD-TH ChR2 mice had several crosses to platform area similar to WT mice (Fishers LSD, WT vs. 3xTgAD: t(32) = 3.341, P = 0.0021; WT vs. AD-TH eYFP: \u0026nbsp;t(32) = 2.876, P = 0.0071; WT vs. AD-TH ChR: t(33) = 0.6390, P = 0.7775; \u0026nbsp;AD-TH ChR2 vs. AD-TH eYFP: t(33) = 3.3270, P = 0.0102). \u003cstrong\u003ed\u003c/strong\u003e Percent time in the target quadrant (One-way ANOVA, F(3,32) \u0026nbsp;= \u0026nbsp;6.912, P = 0.001) showed that 3xTgAD and AD-TH eYFP have a reduced time in the target quadrant compared with the WT group. The AD-TH ChR2 group showed a percentage time in the quadrant similar to WT and better performance than AD-TH eYFP group (Fishers LSD, WT vs. 3xTgAD: t(32) = 3.383, P = 0.0019; WT vs. AD-TH eYFP: \u0026nbsp;t(32) = 2.862, P = 0.0074; WT vs. AD-TH ChR2: t(32) = 0.1874, P = 0.8525; \u0026nbsp;AD-TH ChR2 vs. AD-TH eYFP: t(32) = 2.968, P = 0.0056). \u003cstrong\u003ee\u003c/strong\u003e Latency time to reach the platform area (One-way ANOVA, F(3,32) \u0026nbsp;= \u0026nbsp;3.928, P = 0.0171). As the other parameters, the 3xTgAD and AD-TH eYFP have a more time latency time compared with the WT (Fishers LSD, WT vs. 3xTgAD: t(32) = 2.686, P = 0.0114; WT vs. AD-TH eYFP: \u0026nbsp;t(32) = 2.043, P = 0.0494). The AD-TH groups recovered the LTM, showing less latency time than the WT group, and the performance is better than the AD-TH eYFP group (Fishers LSD, WT vs. AD-TH ChR2: t(32) = 0.0525, P = 0.9584; \u0026nbsp;AD-TH ChR2 vs. AD-TH eYFP: t(32) = 2.048, P = 0.0489). \u003cstrong\u003ef \u003c/strong\u003eSwimming speed. The mean swimming speed was measured to discard any motor deficit that could influence the results. No statistical differences were observed across the mice groups (Mean speed: One-way ANOVA, F(3,32) \u0026nbsp;= \u0026nbsp;1.425, P = 0.2537). The blue bars indicate the optogenetic stimulation. All results showed the mean ± SEM. *: P \u0026lt; 0.05 comparted with WT, $: P \u0026lt; 0.05 comparted with eYFP. HPC: Hippocampus.\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-5868268/v1/127e66bfefcb987560ab0b2d.png"},{"id":74449807,"identity":"fd8fe014-ec1f-4bc5-b8de-ccb935b6ba5b","added_by":"auto","created_at":"2025-01-22 11:41:25","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":7519722,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOptogenetic stimulation of the catecholaminergic LC-CA1 projection restores the synaptic plasticity in AD-TH mice. a\u003c/strong\u003e Schematic representation of bilateral injection of the adenoviral vector carrying ChR2 or eYFP proteins in the LC of AD-TH mice. \u003cstrong\u003eb\u003c/strong\u003e The diagram depicts the optic fiber, recording electrode, and stimulation electrode in the Schaffer collaterals in the CA1 hippocampus. \u003cstrong\u003ec\u003c/strong\u003e The high-frequency stimulation (HFS) protocol involved three trains of 100 pulses at 100 Hz. \u003cstrong\u003ed\u003c/strong\u003e fEPSP recordings in WT (n=8) mice before and after HFS. \u003cstrong\u003ee\u003c/strong\u003e fEPSP recordings in 3xTgAD (n=9) mice before and after HFS.\u003cstrong\u003e f \u003c/strong\u003efEPSP recordings in eYFP AD-TH (n=4) mice before and after HFS. \u003cstrong\u003eg \u003c/strong\u003efEPSP recordings in ChR2 AD-TH (n=9) mice before and after HFS. \u003cstrong\u003eh\u003c/strong\u003e Bar graph of the mean fEPSP recording during the last fifteen minutes of measurements. One-way ANOVA: F(3,24) = 7.673, P \u0026lt; 0.0009; Holm-Sidaks, WT vs. 3xTgAD: t(24) = 3.132, P = 0.018; WT vs. AD-TH eYFP: t(24) = 2.838, P = 0.027; WT vs. AD-TH ChR2: t(24) = 0.819, P = 0.6646; AD-TH eYFP vs AD-TH ChR2: t(24) = 3.449, P = 0.0104). The blue bar represents the optogenetic stimulation (20 Hz, 5 ms, 473 nm to 10-15 mW), and the arrow indicates the HFS. All results show the mean of %fEPSP slope\u003cstrong\u003e \u003c/strong\u003eof\u003cstrong\u003e \u003c/strong\u003ebaseline ± SEM. *: P \u0026lt; 0.05 compared with WT, and $: P \u0026lt; 0.05 compared with AD-TH eYFP.\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-5868268/v1/55f79a42e8614d9403c0ccc1.png"},{"id":74449795,"identity":"be012252-6d4c-4d3b-b91b-8eaeaacd5aa5","added_by":"auto","created_at":"2025-01-22 11:41:24","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":5755461,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOptogenetic stimulation of the catecholaminergic LC-CA1 projection restores the catecholaminergic levels in AD-TH mice.\u003c/strong\u003e \u003cstrong\u003ea\u003c/strong\u003e Schematic representation of bilateral adenoviral injection of AAV-ChR2 or AAV-eYFP in the LC of AD-TH mice. \u003cstrong\u003eb\u003c/strong\u003e Diagram showing the guide cannula and the optic fiber implantation in the CA1 hippocampus.\u003cstrong\u003e c\u003c/strong\u003e Neurotransmitter sample collection protocol before (BASAL fraction), during (STIM fraction, optogenetic stimulation at 20 Hz, 5 ms, 473 nm to 10-15 mW), and after (POST1 and POST2 fractions) the optogenetic stimulation of the LC catecholaminergic projections into CA1 hippocampus. \u003cstrong\u003ed\u003c/strong\u003e Percent of change of NA levels relative to WT mice levels. \u0026nbsp;Compared to WT mice (n=10; NA (One-way ANOVA: F(9,86) = 3.105, P = 0.0028), the analysis revealed that basal levels of \u0026nbsp;NA \u0026nbsp;in the \u0026nbsp;AD models are reduced (3xTgAD 32.83% ± 5.76%, AD-TH eYFP 35.08% ± 9.40%, AD-TH ChR2 35.34% ± 18.46%) (Fisher’s LSD, WT vs. 3xTgAD: t(86) = 2.778, P = 0.0067; WT vs. AD-TH eYFP: t(86) = 2.508, P = 0.0140; WT vs. AD-TH ChR2 t(86) = 2.441, P = 0.0167). \u0026nbsp;The optogenetic stimulation of the LC catecholaminergic projections into CA1 hippocampus augmented the NA levels (Fisher’s LSD, AD-TH eYFP vs. AD-TH ChR2 t(86) = 2.778, P = 0.0299) and after the optogenetic stimulation remain highest (POST1; AD-TH ChR2: 108.0% ± 47.29%; AD-TH eYFP: 20.04% ± 7.07%) (Fisher’s LSD, AD-TH eYFP vs. AD-TH ChR2 t(86) = 3.129, P = 0.0024) \u003cstrong\u003ee\u003c/strong\u003e Percent of change of DA levels relative to WT mice levels. \u0026nbsp;Basal levels are reduced compared with WT mice (3xTgAD 17.81% ± 4.225%, AD-TH eYFP 19.66% ± 2.11%, AD-TH ChR2 16.74% ± 2.83%) (Fisher’s LSD, WT vs. 3xTgAD: t(96) = 4.740, P \u0026lt; 0.0001; WT vs. AD-TH eYFP: t(96) = 4.329, P \u0026lt; 0.0001; WT vs. AD-TH ChR2: t(96) = 4.383, P \u0026lt; 0.0001). \u0026nbsp;The optogenetic stimulation of the LC catecholaminergic projections into CA1 hippocampus augmented DA levels during optogenetic stimulation (Fisher’s LSD, AD-TH eYFP vs. AD-TH ChR2 t(96) = 2.492, P = 0.0144) and after the optogenetic stimulation remains highest (POST 1; Fisher’s LSD, AD-TH eYFP vs. AD-TH ChR2 t(96) = 2.649, P = 0.0094) (POST2; Fisher’s LSD, AD-TH eYFP vs. AD-TH ChR2 t(96) = 3.020, P = 0.0032). The blue bar represents the optogenetic stimulation. All results showed the mean of % of change relative to WT ± SEM. *: p \u0026lt; 0.05 indicated the statistically significant difference compared to WT, and $: p \u0026lt; 0.05 indicated the statistically significant difference related to own eYFP.\u003c/p\u003e","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-5868268/v1/ff83b0059fafce324210840d.png"},{"id":74449808,"identity":"0623b7b3-520f-4627-8e7d-bb2f27519726","added_by":"auto","created_at":"2025-01-22 11:41:26","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":89929523,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePhenotypical characterization of the AD-TH mice\u003c/strong\u003e. Confocal images of CA1 hippocampal coronal sections showing the immunoreactive signal for \u003cstrong\u003ea\u003c/strong\u003e Aβ protein (green), \u003cstrong\u003eb \u003c/strong\u003eHyperphosphorylated tau protein (green), \u003cstrong\u003ec\u003c/strong\u003e TH(red), and DAPI (blue) in aging WT (n=4), 3xTgAD (n=4) and AD-TH (n=4) mice. The ratio of the immunoreactive signal adjusted to the WT \u0026nbsp;mice signals for \u003cstrong\u003ed\u003c/strong\u003e Aβ protein (one-way ANOVA F: (2,9) = 132.5, p \u0026lt; 0.0001; WT vs. 3xTgAD: p \u0026lt; 0.0001; WT vs. AD-TH p \u0026lt; 0.0001; AD-TH vs. 3xTgAD: p = 0.1274) \u003cstrong\u003ee\u003c/strong\u003e Hyperphosphorylated tau protein (one-way ANOVA F: (2,9) = 136.0, P \u0026lt; 0.0001; WT vs. 3xTgAD: p \u0026lt; 0.0001; WT vs. AD-TH, p \u0026lt; 0.0001; AD-TH vs. 3xTgAD: p = 0.8163) and\u003cstrong\u003e f\u003c/strong\u003e THprotein (one-way ANOVA F: (2,9) = 7.388, p = 0.0126; WT vs. 3xTgAD: p = 0.0488; WT vs AD-TH: p = 0.0127; 3xTgAD vs AD-TH: p = 0.6712) within CA1 hippocampus. The second-panel set displays confocal images of LC coronal sections showing the immunoreactive signal for \u003cstrong\u003eg\u003c/strong\u003e Aβ protein (green), \u003cstrong\u003eh\u003c/strong\u003eHyperphosphorylated tau protein (green), \u003cstrong\u003ei \u003c/strong\u003eTH (red), and DAPI (blue) in 12 months aging WT n=4), 3xTgAD (n=4) \u0026nbsp;and AD-TH (n=4) mice. The ratio of the immunoreactive signal adjusted to the WT \u0026nbsp;mice signals for \u003cstrong\u003ej\u003c/strong\u003e Aβ protein (one-way ANOVA, F: (2,9) = 20.61, p= 0.0004; WT vs. 3xTgAD: p = 0.0005; WT vs AD-TH p = 0.0027; AD-TH vs. 3xTgAD: P = 0.4008), \u003cstrong\u003ek \u003c/strong\u003eHyperphosphorylated tau protein (one-way ANOVA F: (2,9) = 130.5, P \u0026lt; 0.0001; WT vs. 3xTgAD: P \u0026lt; 0.0001 WT vs AD-TH P \u0026lt; 0.0001; AD-TH vs. 3xTgAD: P = 0.3096) and \u003cstrong\u003el\u003c/strong\u003e TH protein TH, one-way ANOVA, F: (2,9) = 0.4468, P = 0.6531) in LC. All results show the mean ratio of immunoreactive signal adjusted to WT ± SEM. *: p \u0026lt; 0.05 indicated the statistically significant difference compared to WT\u003cstrong\u003e. \u003c/strong\u003eScale bar: 50 µm.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-5868268/v1/333b8fce0fec4b6a98c0bcb3.png"},{"id":74449788,"identity":"888fa55a-84ed-43e1-bd6c-56cd273a388a","added_by":"auto","created_at":"2025-01-22 11:41:24","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":511445,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Information\u0026nbsp;\u003c/p\u003e","description":"","filename":"SupplementaryInformation.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5868268/v1/011038ce21a586f08e4a6082.pdf"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003ePhotostimulation of Locus Coeruleus CA1 catecholaminergic terminals reversed spatial memory impairment in an Alzheimer's disease mouse model.\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAlzheimer's disease (AD) is characterized by the accumulation of amyloid beta (Aβ) peptides in extracellular aggregates and the formation of intracellular neurofibrillary tangles composed of Tau protein (O\u0026rsquo;Brien and Wong 2011; Sadigh-Eteghad et al. 2015; Zhang et al. 2018; Hampel et al. 2021). The build-up of Aβ peptide and hyperphosphorylated Tau protein tangles in different brain parts has been shown to disrupt the neurotransmitter system, synaptic plasticity, and cognitive function in laboratory and clinical studies (Tolar et al. 2021; Takahashi et al. 2021; Gloria et al. 2021; Wang et al. 2024). However, despite being the leading indicator of this neurodegenerative disease, a direct mechanism explaining the cognitive decline has not been established (Ratan et al. 2023). Despite numerous attempts to address these pathological hallmarks through clinical approaches, developing effective treatments for AD remains challenging (Kuo and Rajesh 2019; Passeri et al. 2022). This emphasizes the urgent need for a deeper understanding of the mechanisms behind the cognitive decline in AD (Ratan et al. 2023).\u003c/p\u003e \u003cp\u003eThe hippocampus plays a crucial role in processing spatial memory and is significantly affected in AD patients from the early stages (Philippen et al. 2024; Billaud and Yu 2024). Initial investigations of post-mortem AD brains have revealed significant disturbances in the hippocampal structure and connectivity, including catecholaminergic projections from the Ventral Tegmental Area (VTA) and Locus Coeruleus (LC) (Sala et al. 2021; Dai et al. 2023). Additionally, AD murine models show diminished catecholaminergic innervation within the hippocampus, correlating with alterations in neuronal excitability and modified gamma-wave oscillatory activity (Nobili et al. 2017; Sakai et al. 2023; Spoleti et al. 2024). Disturbances of the catecholaminergic system, such as decreased catecholamine neurotransmitter levels (Pan et al. 2020) and quantities of catecholamine receptors (Kemppainen et al. 2003; Szot et al. 2006), are common in AD. Damage to catecholaminergic neurons has been linked to memory deficits in animal models of AD and may contribute to the initial symptoms experienced by AD patients (Gloria et al. 2021; Iannitelli et al. 2023; Bueichek\u0026uacute; et al. 2024). Moreover, several studies have suggested the crucial role of catecholaminergic transmission in improving memory deficits in AD. Pharmacological activation of the catecholaminergic system through the use of dopamine (DA) agonists, the DA precursor L-DOPA, or the monoamine oxidase-B inhibitor selegiline has been shown to enhance synaptic plasticity, increase dendritic spine density, modulate hippocampal postsynaptic composition, and improve memory deficits in experimental AD models (Swanson-Park et al. 1999; Yuan Xiang et al. 2016; Moreno-Castilla et al. 2016; T\u0026aacute;bi et al. 2020; Tsetsenis et al. 2022; Alborghetti et al. 2024; Basir et al. 2024).\u003c/p\u003e \u003cp\u003eRecent research has shifted focus from the VTA to the LC as a significant catecholaminergic neuromodulatory structure in the hippocampal circuitry for memory modulation. The LC, which sends denser catecholaminergic inputs to CA1 than the VTA, has been found to modulate synaptic plasticity and spatial memory processes (Takeuchi et al. 2016; James et al. 2021; G\u0026aacute;lvez-M\u0026aacute;rquez et al. 2022). In the early stages of AD, damage and degeneration of the catecholaminergic neurons emerge as a direct consequence of high energy expenditure and large oscillations on intracellular Ca2+, leading to oxidative stress. Furthermore, the DA can be auto oxidized, producing dopaquinone and reactive oxygen species. This oxidative stress increases proinflammatory cytokine expression, disrupting synaptic efficiency (T\u0026ouml;nnies and Trushina 2017). Additionally, the α2A adrenergic receptors, which are highly expressed in the LC, play a role in developing tau tangles through the GSK3β/tau signaling cascade (Braak and Del Tredici 2004; Ferrari et al. 2006; Khaliq and Bean 2010; Surmeier et al. 2017; Zhang et al. 2020; Matchett et al. 2021; Guzm\u0026aacute;n-Ramos et al. 2022). These molecular events and the characteristics of catecholaminergic neurons contribute to reduced LC innervation to other brain structures. This has been linked to decreased noradrenaline (NA) and DA levels in AD patients and mouse models, correlating with memory decline (Kelly et al. 2017). However, it is unclear whether restoring LC catecholaminergic transmission could alleviate the memory deficits associated with AD.\u003c/p\u003e \u003cp\u003eTo study the effects of stimulating LC catecholaminergic transmission on hippocampal synaptic plasticity and spatial memory retrieval, we selectively stimulated the catecholaminergic projections from the LC into the CA1 region of the dorsal hippocampus in a modified 3xTg-AD mouse model (AD-TH), which expresses the CRE recombinase protein under the control of the tyrosine hydroxylase (TH) promoter. We used optogenetic stimulation to activate the LC catecholaminergic projections into the CA1 hippocampal region before the retrieval session in two different spatial memory protocols: the Morris Water Maze (MWM) and Object Location Memory (OLM). Our findings indicate that optogenetic stimulation of the LC catecholaminergic projections into the CA1 hippocampal region before memory retrieval effectively reversed memory impairment in AD mice.\u003c/p\u003e \u003cp\u003eFurthermore, we propose that the spatial memory retrieval deficit in AD-TH mice is partly due to decreased basal levels of catecholamines in the CA1 hippocampal region, which is associated with a reduced number of LC catecholaminergic projections and deficient synaptic plasticity in the same hippocampal region in aging. Interestingly, optogenetic stimulation of these LC catecholaminergic projections restored the levels of catecholamines and synaptic plasticity in the CA1 hippocampal region in these mice. These findings suggest potential therapeutic implications for targeting LC catecholaminergic pathways in AD-related cognitive decline.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eSubjects\u003c/h2\u003e \u003cp\u003eC57BL/6J wild-type mice (WT), along with 3xTgAD and TH-Cre x 3xTgAD (AD-TH) transgenic male and female mice, aged 3 (young) and 12 months (aging), were used during the optogenetic, electrophysiological, and behavioral experiments. The AD-TH strain was created by breeding 3xTgAD mice (Guzm\u0026aacute;n-Ramos et al. 2012; Moreno-Castilla et al. 2016) with TH-Cre mice (G\u0026aacute;lvez-M\u0026aacute;rquez et al. 2022). Genotyping was performed to confirm the presence of the presenilin 1, amyloid precursor protein (APP), human tau, and Cre genes in the offspring. These mice were selectively bred for at least four generations alongside 3xTgAD mice to establish a strain expressing presenilin 1, APP, and tau proteins, with Cre-recombinase regulated by the TH promoter. For genotyping, we used the hotshot method. A tail snip was lysed in an alkaline reagent of 25 mM NaOH and 0.2 mM disodium EDTA at 95\u0026deg;C for 1 hour. After lysis, the DNA was neutralized using 1 M Tris-HCl (pH 7.4) and centrifugated at 2500 rpm for 2 minutes in a Hermie Z 233 MK-2 centrifuge. Finally, the DNA supernatant was collected. This supernatant was amplified using PCR (specific protocol number from QIAGEN) and analyzed via agarose gel electrophoresis.\u003c/p\u003e \u003cp\u003eThe mice were acquired from the Institute of Cellular Physiology animal facility at the National Autonomous University of Mexico. They were individually housed in transparent acrylic cages with ad libitum access to food and water, maintained under a 12/12-hour light/dark cycle at a temperature of 22\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C and relative humidity of 50\u0026thinsp;\u0026plusmn;\u0026thinsp;5%. All experimental procedures were conducted during the light phase of the cycle, and ethical approval was obtained from the Institutional Animal Care and Use Committee of the Institute of Cellular Physiology (Approval No. FBR125-18), adhering to the guidelines outlined in the Official Mexican Standard (NOM-062-ZOO-1999).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eStereotaxic surgery\u003c/h3\u003e\n\u003cp\u003eAll animals were initially anesthetized using a mixture of oxygen (1L per min) and isoflurane (induction 5%; Maintenance 1\u0026ndash;2%; Vip 3000 matrix) before being secured in a stereotaxic apparatus (RWM Life Science, Texas, USA). Surgical coordinates were referenced from the Allen Brain (Atlas Allen Institute for Brain Science). For optogenetic experiments, AD-TH mice were bilaterally injected with either Channelrhodopsin (ChR2) or empty viral vectors. Specifically, the ChR2 group received 0.5 \u0026micro;L per hemisphere of rAAV5/EfIα-DIO-hChR2 (viral concentration of 5.2 \u0026times; 10\u003csup\u003e12\u003c/sup\u003e virus molecules/ml), while the control group received 0.5 \u0026micro;L per hemisphere of rAAV5/EfIα-DIO-eYFP (with a viral concentration of 6.0 \u0026times; 10\u003csup\u003e12\u003c/sup\u003e virus molecules/ml, where eYFP stands for enhanced yellow fluorescent protein). The injections were targeted to the locus coeruleus (LC) at coordinates \u0026minus;\u0026thinsp;5.5 mm anterior-posterior (AP), \u0026plusmn;\u0026thinsp;0.9 mm medial-lateral (ML), and \u0026minus;\u0026thinsp;3.3 mm dorsal-ventral (DV) relative to bregma. The virus was administered using calibrated glass micropipettes (5 \u0026micro;L, Drummond, USA). Additionally, optical fibers were bilaterally implanted in the dorsal hippocampus CA1 at coordinates \u0026minus;\u0026thinsp;2.40 mm AP, \u0026plusmn;\u0026thinsp;2.0 mm ML, and \u0026minus;\u0026thinsp;1.00 mm DV relative to bregma. Mice used for the microdialysis experiments were implanted with an optical fiber (0.22 NA, 200 \u0026micro;m diameter; Doric Lenses, Canada) into the CA1 region of the hippocampus (\u0026minus;\u0026thinsp;2.40 mm AP; \u0026plusmn;2.0 mm ML; \u0026minus;1.00 mm DV to bregma). A cannula guide (CMA/7; CMA Microdialysis, Sweden) was also bilaterally implanted into the CA1 (\u0026minus;\u0026thinsp;3.0 mm AP; \u0026plusmn;2.0 mm ML; \u0026minus;1.5 mm DV to bregma) at a 25\u0026ordm; frontal angle. After surgery, mice were allowed to recover for two weeks before beginning the experiments.\u003c/p\u003e\n\u003ch3\u003eObject Location Memory (OLM) task\u003c/h3\u003e\n\u003cp\u003eThe OLM task was conducted in an open field inside a gray wooden box (33 x 33 x 30 cm) covered with a thin layer of sawdust. A spatial cue was positioned in one of the walls of the box. Two different Lego figures (5 x 5 x 5cm) were used as objects to be explored. Using Debut Video Capture-NCH software version 5.73, a video camera was positioned above the box to record the sessions. Mice were habituated to the box for 10 minutes without any objects for three consecutive days. During the following two acquisition sessions (10 minutes each day), the mice were allowed to explore two objects placed in specific positions. After a 24-hour interval, the long-term memory (LTM) test was conducted. During this test, the mice were allowed to explore the two objects for 10 minutes freely; however, one of the objects was relocated to a novel position for the trial. The novel and familiar positions of the objects were always counterbalanced to prevent any side preference in the arena. The boxes and objects were deodorized with 70% ethanol, and the sawdust bedding was changed between trials. The AD-TH mouse groups (eYFP and ChR2) received optogenetic stimulation (using a 473 nm laser at 10\u0026ndash;15 watts and 20 Hz for 10 minutes) in the hippocampal CA1 region during the LTM test. The sessions were recorded and analyzed offline, focusing on the total exploration time for each object during the acquisition and LTM sessions. The recognition index was calculated by dividing the exploration time for each object by the total exploration time for both objects during each session. It was expected that the mice's recognition index would be close to 0.5 during the acquisition phase, indicating no preference for any object, in order to be included in the statistical analysis.\u003c/p\u003e\n\u003ch3\u003eMorris Water Maze (MWM) task\u003c/h3\u003e\n\u003cp\u003eA MWM behavioral task was conducted in a circular pool (110 cm in diameter) with a white bottom, where an escape platform (15 cm x 15 cm x 15 cm) was positioned at a fixed location 0.5 cm under the water level to remain invisible to the mice. Two geometric figures placed in opposite positions on the pool walls were used as spatial cues. A video camera was placed above the pool to record the trials during the acquisition and the LTM sessions using Debut Video Capture-NCH software version 5.73. Mice were handled for three minutes three days before the acquisition sessions to reduce stress. The acquisition sessions consisted of four training trials daily for four days. Animals were placed at different pre-established starting positions for each trial, with the experimenter always positioned in the same place, serving as a spatial cue. Mice were allowed to swim for 60 seconds and reach the escape platform. The time to reach the platform was recorded; if the mice could not find the escape platform within 60 seconds, the experimenter guided them. Once on the platform, the mice were allowed to explore for 30 seconds. After each trial, each mouse was placed in an open box for 60 seconds to rest before returning to the pool for the subsequent trial.\u003c/p\u003e \u003cp\u003eThe LTM test was conducted 48 hours after the last training session. During the LTM test, the escape platform was removed from the pool, and mice were allowed to swim freely for 60 seconds. AD-TH mouse groups (eYFP and ChR2) were optogenetically stimulated (473nm ser, 10\u0026thinsp;\u0026minus;\u0026thinsp;1,5 watts, 20 Hz, 5 minutes) in the hippocampal CA1 immediately before the LTM test (Kempadoo et al. 2016). Video records were analyzed offline to determine the number of platform crossings, time in the target quadrant, and swimming speed. The video was utilized to determine the area and location of the escape platform during the LTM sessions. The time each mouse spent reaching this area (latency to platform) and the number of times mice crossed to the area (number of crosses) were quantified. For the time analysis in the target quadrant, the pool was divided into four equal quadrants in the video, and the quadrant containing the escape platform was defined as the target quadrant. The swimming time of mice in the target quadrant was quantified. Swimming speed was quantified using the Image J tracking function.\u003c/p\u003e\n\u003ch3\u003eElectrophysiology\u003c/h3\u003e\n\u003cp\u003eThe mice were anesthetized with sodium pentobarbital, initially at a dose of 20 mg/kg of body weight, and then a maintenance dose of 10 mg/kg was administered 60 minutes later. Once anesthetized, the mice were positioned in a stereotaxic apparatus (51603, Stoelting, Chicago, USA). A skin incision was made to expose the skull, and a stainless-steel concentric bipolar stimulation electrode was implanted in the CA3 region of the hippocampus (coordinates from bregma: AP 1.5 mm, ML 2.0 mm, DV 1.0 to 1.5 mm) using a unilateral trepan. A stainless-steel monopolar electrode was placed in the CA1 region of the hippocampus (coordinates from bregma: AP -2.2 mm, ML 1.4 mm, DV 1.0 to 1.5 mm) to record unilateral responses. Stimulation was provided using an AM Systems Model 2100 stimulator and delivered to the stimulation electrode. After establishing a baseline electrically evoked response for 15 minutes with a stimulation intensity set at 50% of the maximum EPSP amplitude, a long-term potentiation (LTP) protocol was initiated. This involved delivering four stimulation trains (1 second at 100 Hz) with 20-second intervals between each train, referred to as high-frequency stimulation (HFS). The evoked responses following this stimulation were recorded for an hour, and field excitatory post-synaptic potentials (fEPSP) were calculated as the percentage change in evoked response amplitude compared to the baseline. AD-TH mouse groups (eYFP and ChR2) were optogenetically stimulated using a 473 nm laser at 10\u0026ndash;15 watts and 20 Hz for 5 minutes before the HFS and 10 minutes after the HFS. Offline analysis was conducted using the fEPSP recorded before and after the HFS.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eMicrodialysis\u003c/h2\u003e \u003cp\u003eA CMA/7 membrane (CMA Microdialysis, Sweden) was inserted into the guide cannula previously implanted in mice. Ringer solution (MgCl2 12mM, NaCl 1.44 M, CaCl2 17 mM and KCl 48 mM) was perfused at a 0.25 \u0026micro;l/min rate using a micro-infusion continuous pump (100 pump CMA Microdialysis, Sweden). Mice were placed into the arena without objects and infused with Ringer solution to collect the neurotransmitter using the EICOM piping system (Concise Freely Moving System, EICOM, USA). Once the 30-minute stabilization period concluded, three 16-minute samples were collected to calculate the baseline. These samples were placed into a vial containing an antioxidant blend (Ascorbic acid 25 mM, Na2EDTA 27 mM, and acetic acid 1 M). Subsequently, a fourth fraction was collected while the mice (eYFP and ChR2) were optogenetically stimulated (473nm laser, 10\u0026ndash;15 watts, 20 Hz, 16 minutes), and two post-stimulation samples were also collected. All samples were stored at -80\u0026ordm;C.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eNeurotransmitter analysis\u003c/h3\u003e\n\u003cp\u003eNeurotransmitter concentration was quantified by capillary electrophoresis. Briefly, all microdialysis samples were prepared for derivatization by adding 6\u0026micro;L of 3-(2- complete)-quinoline-2-carboxaldehyde (FQ, 16.67 mM, Molecular Probes; Massachusetts Invitrogen, USA) and catalyzed with 2\u0026micro;L KCN (24.5 mM) in borate buffer (10 mM pH 9.2). 1 \u0026micro;L of an internal standard (0.075 mM, O-methyl-L-threonine; Fluka, Indiana, USA) was added to each microdialysis sample, and they were incubated in the dark for 15 min at 65\u0026ordm;C. Neurotransmitters were quantified with laser-induced within a capillary electrophoresis system (P/ACE MDQ, Beckman Coulter; Pasadena, USA). Compound separation was based on the micelle electrokinetic chromatography method. Microdialysis samples were hydro-dynamically injected into the capillary system at 0.5 psi for 5s. Separation occurred in a buffer (borates 35 mM, sodium dodecyl sulfate 25 mM, and 13% methanol HPLC grade, pH 9.6) at neurotransmitters was detected by fluorescence using a LIF device (488nm). Signals were depicted as electropherograms and analyzed offline using 32Karat TM8.0 software (Beckman Coulter, Pasadena, USA). Neurotransmitters were identified by comparison with the standard electropherogram pattern (DA and NA standards). Signals were quantified by measuring the ratio of the area under the curve for each neurotransmitter and the area under the curve of its respective internal standard.\u003c/p\u003e\n\u003ch3\u003eImmunofluorescence and confocal microscopy\u003c/h3\u003e\n\u003cp\u003eMice were euthanized utilizing sodium pentobarbital (75 mg/kg) after the behavioral, electrophysiological, and neurotransmitter analysis experiments. Immediately after euthanasia, mice were perfused with a 0.9% saline solution and fixed with a 4% paraformaldehyde solution. The brain was extracted and preserved in a 4% paraformaldehyde solution. A cryostat (Leica CM520, Germany) cut mice brains in 40 \u0026micro;m coronal slices. Free-floating tissue slices were incubated overnight with primary antibodies diluted in a 5% bovine serum albumin buffer (NaCl 150 mM, Triton X-100 0.1%, Trizma base 100 mM, pH 7.4) at a dilution of 1:1000 (mouse monoclonal anti-human phospho-PHF-tau pSer202/Thr205 antibody, Thermo Scientific, Belgium; mouse monoclonal BAM-10 anti-beta-amyloid antibody, Missouri, Sigma Aldrich, USA; rabbit polyclonal anti-TH antibody, Arkansas, Pel-Freez, USA). In the case of immunohistochemistry against Aβ, the floating tissues were incubated for 5 minutes in formic acid and washed with TBS-T 0.1% before the primary antibody incubation. The next day, floating slices were washed 6 times with Tris-buffered saline solution and then incubated with secondary antibodies diluted in 5% bovine serum albumin buffer at a 1:500 dilution (Mouse IgG antibody (FITC), California, GeneTex, USA; Gt X Rb IgG Cy3, Massachusetts, Millipore, USA) for 2 hours. After that, the slices were mounted using a Dako fluorescence mounting medium. The immunofluorescence was examined using a ZEISS LSM 800 confocal microscope (Zeiss, Germany). The immunofluorescence images were digitized for further analysis using Image J (version), and the antibody signal was obtained and quantified by measuring the percentage of the pixel area.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eStatistics\u003c/h2\u003e \u003cp\u003eStatistics analysis was conducted using GraphPad Prism software (version 7.00, USA). All graphs showed mean\u0026thinsp;\u0026plusmn;\u0026thinsp;sem with a statistical significance of p\u0026thinsp;\u0026lt;\u0026thinsp;0.05. For immunohistochemistry, images were processed with Image J software and analyzed with One-way ANOVA and Tukey post hoc. OLM and MWM data were analyzed with one or two-factor ANOVA, followed by multiple comparison tests with statistical significance determined using the Fisher's LSD or Tukey tests. Microdialysis analyses were conducted using one-way ANOVA and Holm-Sidak's post hoc analysis. One-way ANOVA and Holm-Sidak's post hoc analysis were performed for electrophysiology.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eOptogenetic stimulation of the LC-CA1 projections enhances spatial memory retrieval in an AD-TH mouse model of Alzheimer's disease\u003c/b\u003e \u003c/p\u003e \u003cp\u003eResearch has demonstrated the importance of catecholamines in spatial memory, mainly through optogenetic techniques. These studies have shown that inhibiting the catecholaminergic projections from the LC to the hippocampal CA1 region leads to impaired spatial memory expression (G\u0026aacute;lvez-M\u0026aacute;rquez et al. 2022). Notably, a reduction in these projections has been observed in the early stages of AD (Theofilas et al. 2017; James et al. 2021), which motivated us to investigate further the effects of optogenetic activation of the LC projections on hippocampal-dependent memory and synaptic plasticity in a murine model of AD. To achieve this, we used an adenoviral vector to express the ChR2 protein or the enhanced eYFP reporter protein in LC neurons. Additionally, we implanted an optic fiber in the CA1 region of the hippocampus in our modified AD-TH mouse model (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea and b). To evaluate the viral infection efficacy in the AD-TH mice's LC neurons and their hippocampal projections (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea and b), we conducted an immunohistochemical analysis of the eYFP reporter protein. This analysis assessed its expression in LC TH\u0026thinsp;+\u0026thinsp;neurons and their projections within the CA1 hippocampal region. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec, the eYFP reporter protein is expressed in the LC neurons that project to the CA1 hippocampal region. Furthermore, the TH immunoreactive signal colocalizes with the eYFP immunoreactive signal in both brain regions (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec).\u003c/p\u003e \u003cp\u003eWe implemented a behavioral paradigm to assess hippocampal-dependent OLM (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed) and to evaluate the effect of the optogenetic stimulation of the catecholaminergic LC projections within the CA1. During the two-day acquisition sessions, all groups of mice exhibited an equal exploration of the two objects, evidenced by their recognition index, demonstrating no preference to explore either object (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee). Twenty-four hours post-acquisition, the AD-TH mice underwent optogenetic stimulation during a 10-minute LTM session. While WT mice recognized the object in a novel location compared to a familiar one, both 3xTgAD and AD-TH eYFP mice failed to show recognition of the object in the novel location versus the familiar location. This indicates a deficit in retrieving LTM in OLM, likely due to the AD pathology. In contrast, optogenetic stimulation of the catecholaminergic LC projections within CA1 improved the retrieval of OLM in AD-TH mice expressing ChR2, demonstrated by a higher recognition index for the object in the novel location than the familiar location (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ef). Additionally, young AD mice and young AD-TH could retrieve LTM in OLM, evidenced by a preference to explore the object in a novel location vs a familiar location (Supplementary Fig.\u0026nbsp;1a, b, and c). At the same time, no difference in the total exploration time was observed (Supplementary Fig.\u0026nbsp;1d).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eA MWM behavioral protocol was utilized to corroborate the effect of optogenetic activation of the catecholaminergic LC-CA1 projection on the recovery of spatial memory retrieval. Both young and aging mice were included in the behavioral tests. Four training trials were conducted daily over four days during the acquisition phase. Throughout these sessions, no differences were observed in the latency time to reach the hidden platform among young and old WT, 3xTgAD, and AD-TH mice across the training days. By the fourth day of training, all groups of mice showed reduced latency times to reach the hidden platform (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb, and Supplementary Fig.\u0026nbsp;2b). Forty-eight hours later, during the LTM test, young WT, AD-TH, and 3xTgAD groups showed strong spatial memory retrieval (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec, and Supplementary Fig.\u0026nbsp;2c). However, aging AD mice exhibited deficits in spatial memory retrieval compared to their same-aged WT counterparts. The 3xTgAD and AD-TH eYFP mice showed a reduced number of crossings to the platform area (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec), a lower percentage of time spent in the target quadrant (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed), and longer latency times to reach the platform area (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee). These findings indicate a compromised ability to retrieve spatial memory in these groups.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eInterestingly, the AD-TH ChR2 mice exhibited enhanced spatial memory retrieval when optogenetic stimulation of the catecholaminergic projections from the locus coeruleus to the CA1 region was applied just before testing for long-term memory (LTM). The results showed a greater number of area crossings, indicating a recovery effect. These mice also spent a larger percentage of time in the target quadrant and displayed a shorter latency to the platform area compared to the AD-TH eYFP mice, achieving results like those of the WT mice (see Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec, d, and e). Importantly, swimming speed remained consistent across all groups, with no changes resulting from the optogenetic stimulation of the catecholaminergic LC projections in the CA1 hippocampal region (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ef). These findings suggest that while memory acquisition is unaffected in young and aging AD mice, memory retrieval performance in the MWM and OLM tasks is impaired in aging AD mice. However, activating the catecholaminergic LC projections within the CA1 region of the hippocampus enhances spatial memory retrieval, as evaluated through the OLM and MWM paradigms. This evidence supports the notion that deficiencies in spatial memory retrieval are age-dependent, but activating the catecholaminergic LC projections in the CA1 region is sufficient to mitigate these retrieval deficits in older AD mice.\u003c/p\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eOptogenetic stimulation of the LC-CA1 projections enables LTP induction in AD mice\u003c/h2\u003e \u003cp\u003eReports have shown alterations in hippocampal synaptic plasticity in AD mice (Oddo et al. 2003; T\u0026ouml;nnies and Trushina 2017; Brandwein and Nguyen 2019). To assess whether the enhancement of the catecholaminergic LC neurotransmission within the hippocampus improves synaptic plasticity, mice were injected with the adenoviral vector carrying ChR2 or eYFP reporter proteins in the LC (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). Young and aging AD-TH mice were subjected to LTP protocol coupled with the optogenetic stimulation of the catecholaminergic LC projections in the Shaffer collateral pathway of AD mice under anesthesia (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). LTP was induced by applying an HFS (3 trains of 100 pulses at 100Hz) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec) in the CA3 projections. At the same time, the field fEPSP was measured within CA1 of the hippocampus. Interestingly, young AD mice could induce and maintain a strong LTP (Supplementary Fig.\u0026nbsp;3c and e), suggesting that alterations in the hippocampal synaptic plasticity in AD are aging dependent. In aging mice, WT mice exhibited an augmented fEPSP after HFS compared to the baseline recordings (137.5% \u0026plusmn; 12.07%), and these responses persisted for up to one hour (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed). In contrast, 3xTgAD mice failed to induce LTP; instead, a depression in the fEPSP recordings following HFS was observed (85.41% \u0026plusmn; 9.05%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee). Similarly, eYFP AD-TH mice presented a decrement in the synaptic communication post-HFS (78.01.7% \u0026plusmn; 10.35%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ef). However, the optogenetic stimulation of the catecholaminergic LC projections within the CA1 hippocampal region of ChR2 AD-TH mice reinstated the synaptic plasticity within the Schaffer collaterals pathway (152.7% \u0026plusmn; 17.47%) after HFS (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eg). This observed potentiation of the fEPSP in ChR2 AD-TH mice was similar to that in WT mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eh).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThese results indicate that enhancing the catecholaminergic LC neurotransmission within the CA1 hippocampus significantly improves the synaptic plasticity in that specific brain region. Interestingly, this reinstatement of the synaptic plasticity in the hippocampus is associated with the improved ability of AD mice to retrieve spatial memory effectively.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eOptogenetic stimulation of the LC-CA1 projections increases dopamine and noradrenaline levels in AD-TH mice\u003c/h2\u003e \u003cp\u003eResearch has shown that catecholaminergic neurotransmitters in the hippocampus are reduced in AD mouse models and patients (Kelly et al., 2017; Theofilas et al., 2017; Dahl et al., 2023). This raises an important question: does optogenetic stimulation of the catecholaminergic projections from the locus coeruleus to the CA1 region increase DA and NA levels in an AD mouse model? To investigate this possibility, we conducted capillary electrophoresis analysis using an \u003cem\u003ein vivo\u003c/em\u003e microdialysis approach to quantify catecholaminergic neurotransmitter levels. Adenoviral vectors encoding the Channelrhodopsin-2 (ChR2) protein or the enhanced yellow fluorescent protein (eYFP) reporter protein were injected into the locus coeruleus of AD-TH mice. Simultaneously, neurotransmitter samples were collected from the CA1 region of the hippocampus during the optogenetic stimulation process (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea and b).\u003c/p\u003e \u003cp\u003eNeurotransmitter samples were obtained at three different time points: before the stimulation (BASAL), during the stimulation (STIM), and after the stimulation at two intervals (POST1 and POST2) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec). The quantification of NA and DA levels showed a significant reduction in the CA1 hippocampus of 3xTgAD and AD-TH mice compared to WT mice. The optogenetic stimulation of the LC projections to the CA1 hippocampus successfully restored extracellular levels of NA and DA for up to 30 minutes following the stimulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed and e). These findings indicate that DA and NA concentrations are diminished in the hippocampus of AD mice. This reduction and other pathological processes associated with AD contribute to deficits in spatial memory retrieval. Notably, restoring catecholaminergic neurotransmission from the LC to the hippocampus is sufficient to enhance catecholaminergic levels and improve spatial memory retrieval in AD mice.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003ePhenotypical characterization of the AD-TH mice\u003c/h2\u003e \u003cp\u003eThe AD-TH mouse model was developed to optogenetically activate the catecholaminergic LC projections into the CA1 region of the hippocampus by interbreeding 3xTgAD mice with TH-Cre mice. First, genotyping confirms the presence of presenilin 1, APP, human tau, and Cre genes in AD-TH mice. Then, it was necessary to describe the phenotypic expression of hallmark proteins associated with AD, specifically the Aβ protein and hyperphosphorylated tau protein. These proteins are known to be overexpressed and accumulate within both the LC and the hippocampus in the context of AD pathology. In our analysis, we also examined TH protein levels in both brain structures to quantify the catecholaminergic neuron somas in the LC and the catecholaminergic terminals in the hippocampus of aging mice. A significant increase in Aβ protein (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea and d) and hyperphosphorylated tau protein (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb and e) was observed in the 3xTgAD and AD-TH mice within the CA1 hippocampus compared to WT mice. However, no significant differences in the expression of these proteins were found between the two AD mouse models. Additionally, the TH immunoreactive signal in the hippocampus indicated reduced catecholaminergic projections (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec and f).\u003c/p\u003e \u003cp\u003eSimilarly, Aβ (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eg and j) and hyperphosphorylated tau (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eh and k) proteins showed increased immunoreactive signals in the LC of aging 3xTgAD and AD-TH mouse models. While both proteins were overexpressed in the LC, no significant difference in the immunoreactive signal for TH was found in the neuron somas of WT, 3xTgAD, and AD-TH mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ei and l). A comparative analysis of the Aβ protein immunoreactive signals between the CA1 hippocampus region and LC indicates a more significant accumulation of Aβ proteins in the hippocampus compared to the LC (LC vs. HIP: t(\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e)\u0026thinsp;=\u0026thinsp;8.992, p\u0026thinsp;=\u0026thinsp;0.0001). In contrast, the immunoreactivity of hyperphosphorylated tau protein was significantly higher in the LC than in the hippocampus (LC vs. HIP: t(\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e)\u0026thinsp;=\u0026thinsp;3.469, p\u0026thinsp;=\u0026thinsp;0.0133). These findings suggest that the expression and accumulation of Aβ and hyperphosphorylated tau proteins occur differentially. While TH-AD mice exhibit detectable accumulation of both proteins, Aβ primarily accumulates in the CA1 region of the hippocampus. Conversely, hyperphosphorylated tau protein mainly accumulates in the LC of old AD mice. Furthermore, our phenotypic analysis of catecholaminergic neurons in the LC and their projections to the hippocampus implies that the reduction of catecholaminergic neurotransmitter levels in the hippocampus is due to a decline in the catecholaminergic projections rather than a loss of catecholaminergic neurons in the LC.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eAlzheimer's disease progression involves ongoing damage to neuronal function in both the cortical and hippocampus regions. Notably, mice models of Alzheimer's display significant degeneration of catecholaminergic terminals in the hippocampus around 12 months of age. This damage is followed by neuronal degeneration in the LC and the VTA at 18 and 24 months, respectively. This progressive degeneration of catecholaminergic activity is primarily attributed to the deposition of Aβ peptide (Grudzien et al. 2007; Liu et al. 2008; Moreno-Castilla et al. 2016). They also underscore its significance in developing cognitive deficits associated with AD (Braak and Del Tredici 2016; Hansen 2017; Dahl et al. 2023). Recent research suggests a denser catecholaminergic projection from LC into the CA1 of the hippocampus, as opposed to sparse dopaminergic fibers originating from the VTA (Takeuchi et al. 2016; Kempadoo et al. 2016; Tsetsenis et al. 2022; G\u0026aacute;lvez-M\u0026aacute;rquez et al. 2022). It has been reported that projections from the LC are mainly located in the dorsal regions of the hippocampus, which are relevant for memory processes. In contrast, dopaminergic innervation from the VTA targets the ventral areas of the hippocampus (Titulaer et al. 2021; Wilmot et al. 2024). The LC catecholaminergic projections to the hippocampus have been recognized as pivotal in modulating synaptic plasticity and cellular mechanisms underlying memory (Lemon and Manahan-Vaughan 2012; Mravec et al. 2014; Hansen and Manahan-Vaughan 2015; G\u0026aacute;lvez-M\u0026aacute;rquez et al. 2022). Particularly in the CA1 region, this catecholaminergic role is crucial for restoring place cells accountable for spatial details within the hippocampus (Kaufman et al. 2020). These findings emphasize the significance of LC-hippocampus catecholaminergic neuromodulation in regulating hippocampal-dependent memory processes. Therefore, this abnormally low level of LC catecholaminergic projections into the CA1 region provides a pathological substrate for the retrieval deficiencies observed in AD mice. This reduction in LC catecholaminergic projections within the same hippocampal region has been observed in AD patients and murine models (Reinikainen et al. 1988; Chen et al. 2022).\u003c/p\u003e \u003cp\u003e \u003cb\u003eOptogenetic activation of the LC-CA1 projections improves the catecholaminergic neurotransmission within the hippocampus of AD-TH mice\u003c/b\u003e \u003c/p\u003e \u003cp\u003eIn our research, we observed a significant decrease in the baseline levels of DA and NA in the CA1 area of the hippocampus in 3xTg-AD and AD-TH mice. The reduced levels of these neurotransmitters in the hippocampus of AD patients are thought to be part of the cognitive dysregulation that causes AD symptoms(Lyness et al. 2003; Francis et al. 2012; Hagena et al. 2016). Our findings show that the decrease in DA and NA levels in the CA1 region is linked to a reduction in hippocampal CA1 catecholaminergic projections from the LC rather than a loss of LC catecholaminergic neurons, at least in 12-month-old mice. We also showed that optogenetic stimulation of the CA1 hippocampal catecholaminergic projections from the LC is enough to restore hippocampal catecholaminergic levels. The LC is a crucial brain region where abnormal hyperphosphorylated tau protein accumulates in individuals with AD (Mather and Harley 2016; Beardmore et al. 2021). In both of our transgenic mouse models, the levels of hyperphosphorylated tau protein in the LC align with Braak staging and have been detected before the onset of tau pathology in the hippocampus (Grudzien et al. 2007; Braak and Del Tredici 2016). Although we did not observe a decrease in TH\u0026thinsp;+\u0026thinsp;neurons in the LC of our 12-month-old AD mice\u0026mdash;an indication typically associated with mid- to late-stage AD\u0026mdash;the accumulation of abnormal tau protein could still lead to degeneration of catecholaminergic projections. This degeneration may result in reduced basal release of DA and NA in the hippocampus, which are necessary to retrieve OLM (Moreno-Castilla et al. 2016; G\u0026aacute;lvez-M\u0026aacute;rquez et al. 2022).\u003c/p\u003e \u003cp\u003eAdditionally, the Aβ peptide plays a neuromodulatory role in regulating synaptic communication and neurotransmitter release (Karisetty et al. 2020), mediated through direct and indirect interactions with presynaptic proteins that control neurotransmitter vesicle release (Abramov et al. 2009; Russell et al. 2012; Gulisano et al. 2019). However, the accumulation of Aβ peptide has a detrimental effect on neurotransmitter release. The combination of diminished LC catecholaminergic projections and the potential adverse consequences of Aβ peptide accumulation may contribute to the diminished catecholaminergic levels observed here, thereby impeding the hippocampal synaptic efficiency and negatively impacting the retrieval of spatial memories in AD mice. Nevertheless, we show that the pathological low level of catecholamines in the hippocampus can be reversed through the optogenetic stimulation of the LC catecholaminergic projections into the CA1 hippocampal region in the AD-TH mice.\u003c/p\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eOptogenetic stimulation of the LC-CA1 projections reverses spatial memory retrieval deficiencies in AD mice\u003c/h2\u003e \u003cp\u003eResearch on Alzheimer's Disease suggests that the primary challenge lies in retrieving memories rather than storing them (Roy et al. 2016; Jura et al. 2019; Small and Cochrane 2020; Bostancıklıoğlu 2020). Our findings support this hypothesis. We observed that young AD mice can remember and recall spatial information in both tasks. However, aging mice from the 3xTg-AD and AD-TH models struggle to retrieve this information during LTM assessments. We found that stimulating the catecholaminergic projections from the LC-CA1 of the hippocampus before LTM tasks significantly improved memory retrieval in our AD-TH mouse model, and additionally, activating these catecholaminergic projections in the same CA1 region before retrieval enhanced the ability of the mice to recognize new object locations during LTM sessions. These results suggest that AD-TH mice can form and consolidate hippocampal-dependent memories but face challenges in retrieving these memories. Accordingly, we demonstrated that photoinhibition of the LC catecholaminergic projections into the CA1 hippocampus prevents the retrieval of the spatial memory (G\u0026aacute;lvez-M\u0026aacute;rquez et al. 2022), highlighting the importance of the catecholaminergic activity from the LC to retrieve the spatial memory in the hippocampus adequately.\u003c/p\u003e \u003cp\u003eAdministering a DA D1-like receptor antagonist or a beta-adrenergic receptor antagonist before a LTM session in the CA1 region of the hippocampus impairs spatial memory retrieval. However, DA activity\u0026mdash;rather than NA activity\u0026mdash;is crucial for updating spatial memory (G\u0026aacute;lvez-M\u0026aacute;rquez et al. 2022). In this context, administering NA precursors or DA agonists has been shown to reduce the toxic effects of Aβ and improve performance in spatial memory tasks (Himeno et al. 2011; Kalinin et al. 2012; Guti\u0026eacute;rrez et al. 2022). Research also indicates that blocking DA reuptake in the cortex of AD transgenic mice can alleviate memory recognition impairment and enhance dopaminergic activity (Guzm\u0026aacute;n-Ramos et al. 2012, 2022; Moreno-Castilla et al. 2016). Together, these studies emphasize that catecholaminergic activity is closely related to AD dysfunction, representing a potential neurochemical target for pharmacological treatment. Consequently, using DA agonists, rotigotine, the DA precursor L-DOPA or the monoamine oxidase-B inhibitor selegiline has enhanced synaptic plasticity and improved memory deficits in experimental AD models and humans (Koch et al. 2014; Dahl et al. 2023). Although further investigation is needed to identify the specific catecholaminergic receptors involved in memory retrieval, these findings suggest that stimulating catecholaminergic pathways in the brain could help restore memory retrieval in 12-month-old AD-TH mice. Additionally, these results align with other studies indicating that Alzheimer's patients often struggle with memory recall due to difficulties in activating memory patterns. Notably, this study is the first to highlight the critical and sufficient role of LC catecholaminergic activity in the hippocampus for enhancing memory retrieval in models of Alzheimer's disease.\u003c/p\u003e \u003cp\u003e \u003cb\u003eOptogenetic activation of the LC-CA1 projections restores synaptic plasticity in AD-TH mice.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eOur experimental approach indicates that enhancing spatial memory retrieval in early-stage AD mice may be linked to promoting hippocampal neural plasticity. This improvement could result from restoring catecholaminergic levels through optogenetic stimulation of the LC catecholaminergic projections within the CA1 region of the hippocampus. It is well known that DA and NA's transient bursts (phasic activity) occur in response to rewarding or novel stimuli to promote LTM (Harley 2004). Accordingly, younger AD mice demonstrate normal synaptic plasticity, but by age, they can no longer induce LTP following high-frequency stimulation. These findings suggest that the age of the AD mice influences the inability to enable synaptic plasticity.\u003c/p\u003e \u003cp\u003eCatecholamines play a crucial role in modulating the molecular and cellular mechanisms that contribute to synaptic plasticity through their specific receptors (Twarkowski and Manahan-Vaughan 2016; Madadi Asl et al. 2019). A reduction in innervation can lead to disinhibition and increased excitability in hippocampal areas, often observed in the early stages of Alzheimer's disease (Goettemoeller et al. 2024). NA interacts with α1, α2, and β adrenergic G-protein coupled receptors (GPCRs), activating Gs subunits and cAMP pathways significantly influencing synaptic plasticity (Marzo et al. 2009; Maity et al. 2020, 2022). Additionally, NA can induce hyperpolarization in cortical neurons (Wong et al. 2023) and enhance LTP in the CA1 region by promoting protein kinase A (PKA) pathways (Gelinas et al. 2008) and activating guanine nucleotide exchange proteins through cyclic adenosine monophosphate (cAMP)(Brandwein and Nguyen 2019).\u003c/p\u003e \u003cp\u003eDA influences synaptic function through D1-like and D2-like G protein-coupled receptors GPCRs, which promote LTP and LTD in the hippocampus, respectively (Caragea and Manahan-Vaughan 2021; Kim et al. 2022). While ionotropic receptors mediate rapid neuronal responses through the immediate flow of ions across the cell membrane, GPCRs trigger a slower activation of intracellular signaling cascades. This process indirectly affects ion and neurotransmitter release, ultimately influencing gene expression and synaptic plasticity (Betke et al. 2012). Although hippocampal CA1 synaptic plasticity in the AD-TH mice was reinstated due to optogenetic stimulation of the LC catecholaminergic projections, we observed a slow onset in the induction of LTP. As mentioned earlier, this slow onset may be attributed to the delayed response elicited by the GPCRs (Greengard 2001; Wong et al. 2023; Tse et al. 2023).\u003c/p\u003e \u003cp\u003eRecent studies indicate that synaptic plasticity in the hippocampus can be influenced by optogenetic stimulation of catecholaminergic projections in healthy mice (Takeuchi et al. 2016; Kempadoo et al. 2016; G\u0026aacute;lvez-M\u0026aacute;rquez et al. 2022). The activity of catecholamines from the LC highlights the importance of neural communication within the hippocampus (Hansen 2017). Recent findings demonstrate that photoinhibition of hippocampal catecholamine projections from the LC can alter the threshold for transitioning from LTP to LTD following high-frequency stimulation (G\u0026aacute;lvez-M\u0026aacute;rquez et al. 2022). This change may be linked to decreased levels of DA and NA. Additionally, DA depletion caused by Aβ peptide has been shown to impair synaptic plasticity, converting high-frequency stimulation-induced LTP into LTD in the dorsal hippocampus (Mayordomo-Cava et al. 2020) and cortical circuits (Moreno-Castilla et al. 2016). Similarly, the co-administration of DA and beta-adrenergic receptor antagonists can induce LTD following high-frequency stimulation (G\u0026aacute;lvez-M\u0026aacute;rquez et al. 2022). Interestingly, we have found that increased cortical DA activity can reverse LTD induced by Aβ1\u0026ndash;42 oligomers back into LTP (Guzm\u0026aacute;n-Ramos et al. 2012, 2022; Moreno-Castilla et al. 2016). These results align with the observed rise in catecholamines when photostimulation of the LC-CA1 terminals leads to improved memory and a shift from LTD to LTP in the hippocampal CA1 region. Our experimental approach suggests stimulating the catecholaminergic system is vital for forming LTM by enhancing synaptic plasticity mechanisms. These changes are essential for activating and triggering the memory engram, enabling AD-TH mice to retrieve previously acquired spatial information.\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe exact causes and underlying mechanisms of Alzheimer\u0026rsquo;s disease are not yet fully understood. However, it is essential to develop strategies to improve the memory deficits associated with AD for clinical treatments. Some experimental approaches suggest that stimulating the catecholaminergic system through pharmacological methods (Koch et al. 2014; Shaikh et al. 2023) or repeated exposure to novel stimuli could be critical for cognitive improvement (Vel\u0026aacute;zquez-Delgado et al. 2024). These strategies emphasize the influence of lifestyle and environmental factors on AD and their potential to alleviate symptoms. Research indicates that abnormal tau aggregation in the LC and dysfunction of the catecholaminergic system may represent the early stages of AD progression. Our findings suggest that therapies targeting the projections from the LC to the cortex and hippocampus could enhance cognitive function in patients with mild cognitive impairment due to early AD. These results highlight the significance of targeting LC catecholaminergic neurons as potential therapeutic targets to address cognitive deficits in AD patients through targeted stimulation.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eAD, Alzheimer\u0026acute;s disease.AD-TH, Alzheimer\u0026acute;s disease-Tyrosine hydroxylase\u003cstrong\u003e.\u0026nbsp;\u003c/strong\u003eAPP, Amyloid precursor protein.A\u0026beta;, Beta-amyloid peptide.\u0026nbsp;ChR2,\u0026nbsp;Channelrhodopsin.\u0026nbsp;eYFP,\u0026nbsp;DA,\u0026nbsp;Dopamine\u003cstrong\u003e.\u0026nbsp;\u003c/strong\u003eEnhanced\u0026nbsp;yellow\u0026nbsp;fluorescent\u0026nbsp;protein.EPSP, Excitatory post-synaptic potentials.\u0026nbsp;LC, Locus Coeruleus.\u0026nbsp;LTM, Long-Term memory.LTP, Long-Term potentiation.\u0026nbsp;MWM, Morris Water Maze. NA, Noradrenaline.OLM, Object Location Memory\u003cstrong\u003e.\u0026nbsp;\u003c/strong\u003eTH, Tyrosine hydroxylase\u003cstrong\u003e.\u0026nbsp;\u003c/strong\u003e3Tg-AD, Triple transgenic Alzheimer mouse model.\u0026nbsp;VTA, Ventral Tegmental Area. WT, Wild type.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments and Funding\u003c/strong\u003e\u003cstrong\u003e.\u0026nbsp;\u003c/strong\u003eWe would like to express our gratitude to the Unidad de Imagenolog\u0026iacute;a at the Instituto de Fisiolog\u0026iacute;a Celular, UNAM, particularly to Dr. Ruth Rinc\u0026oacute;n Heredia and Dr. Abraham Rosas Arellano, for their invaluable technical support. We also thank Psychologist Ana Cecilia L\u0026oacute;pez Sep\u0026uacute;lveda for her assistance in conducting the behavioral and electrophysiological experiments. This study was performed as part of the requirements requested to obtain a doctoral degree in Biochemical Sciences for Donovan K. G\u0026aacute;lvez-M\u0026aacute;rquez at UNAM. D.K.G.-M. received the fellowship 856256 from Consejo Nacional de Humanidades Ciencia y Tecnolog\u0026iacute;a (CONAHCYT), M\u0026eacute;xico. This project was supported by the CONAHCyT grants FOINS 474, CF-2023-I-189, and DGAPA-PAPIIT-UNAM grant IN 213123 to F.B.-R.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003cstrong\u003e.\u0026nbsp;\u003c/strong\u003eD.K.G.-M.,O.U.-M., L.R.-D., and F.B.-R. designed research; D.K.G.-M. and O.U.-M. performed research; D.K.G.-M., O.U.-M., L.R.-D., and F.B.-R. \u0026nbsp;analyzed data; and D.K.G.-M.,O.U.-M. and F.B.-R. \u0026nbsp;wrote the paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest disclosure.\u0026nbsp;\u003c/strong\u003eThe authors declare no conflicts of interest.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003cstrong\u003e.\u003c/strong\u003eAnalyzed data will be made available upon reasonable request to the corresponding author.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical Approval\u003c/strong\u003e\u003cstrong\u003e.\u003c/strong\u003e All experimental procedures were carried out in accordance with the ethical approval from the Institutional Animal Care and Use Committee of the Institute of Cellular Physiology (Approval No. FBR125-18), following the guidelines stated in the Official Mexican Standard (NOM-ZOO-1999).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAbramov E, Dolev I, Fogel H et al (2009) Amyloid-β as a positive endogenous regulator of release probability at hippocampal synapses. 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Front Aging Neurosci 10:359. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3389/fnagi.2018.00359\u003c/span\u003e\u003cspan address=\"10.3389/fnagi.2018.00359\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[{"identity":"2e22adf0-be7e-485e-be5f-509a1e83d54e","identifier":"10.13039/501100003141","name":"Consejo Nacional de Ciencia y Tecnología","awardNumber":"FOINS 474, CF-2023-I-189","order_by":0},{"identity":"c32184db-43e2-45f2-8906-f734a134e7fc","identifier":"10.13039/501100006087","name":"Dirección General de Asuntos del Personal Académico, Universidad Nacional Autónoma de México","awardNumber":"IN 213123","order_by":1},{"identity":"4359943e-ea43-40e0-b5d2-f105650dd538","identifier":"10.13039/501100003141","name":"Consejo Nacional de Ciencia y Tecnología","awardNumber":"856256","order_by":2}],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"Universidad Nacional Autónoma de México","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":false,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Dopamine, Alzheimer`s disease, Locus Coeruleus, Hippocampus, Cognitive impairment","lastPublishedDoi":"10.21203/rs.3.rs-5868268/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5868268/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eRationale\u003c/strong\u003e One of the earliest changes associated with Alzheimer's disease (AD) is the loss of catecholaminergic terminals in the cortex and hippocampus originating from the Locus Coeruleus (LC). This decline leads to reduced catecholaminergic neurotransmitters in the hippocampus, affecting synaptic plasticity and spatial memory. However, it is unclear whether restoring catecholaminergic transmission in the terminals from the LC may alleviate the spatial memory deficits associated with AD.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eObjectives\u003c/strong\u003e This study investigates how optogenetic stimulation of catecholaminergic projections from the locus coeruleus to the hippocampal CA1 region may enhance spatial memory and alleviate synaptic plasticity deficits associated with Alzheimer's disease.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e We conducted experiments using a 12-month-old 3xTg-AD mouse model (AD-TH), which expresses Cre recombinase under the control of the tyrosine hydroxylase (TH) gene. This model allowed us to photostimulate the terminals from the locus coeruleus in the hippocampal CA1 region before performing two different spatial memory tasks and inducing long-term potentiation (LTP).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e Optogenetic stimulation successfully reversed the impairment of spatial memory retrieval in aging AD-TH mice. Furthermore, this stimulation restored catecholaminergic neurotransmitter levels in the hippocampus and enhanced synaptic plasticity, as demonstrated by an LTP protocol.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions\u003c/strong\u003e These findings indicate that the catecholaminergic circuitry from the locus coeruleus (LC) to the hippocampal CA1 region plays a crucial role in disrupting synaptic plasticity and contributing to the spatial memory deficits seen in the early stages of AD. This study highlights the potential therapeutic benefits of targeting LC catecholaminergic neurons to improve cognitive function in patients with AD.\u003c/p\u003e","manuscriptTitle":"Photostimulation of Locus Coeruleus CA1 catecholaminergic terminals reversed spatial memory impairment in an Alzheimer's disease mouse model.","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-01-22 11:41:19","doi":"10.21203/rs.3.rs-5868268/v1","editorialEvents":[{"type":"communityComments","content":3}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d8bc229a-530b-4c37-a67c-9aa885894d47","owner":[],"postedDate":"January 22nd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":43150214,"name":"Cognitive Neuroscience"},{"id":43150215,"name":"Neurobiology of Disease"}],"tags":[],"updatedAt":"2025-01-22T11:41:19+00:00","versionOfRecord":[],"versionCreatedAt":"2025-01-22 11:41:19","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5868268","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5868268","identity":"rs-5868268","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}