Chronic Pb exposure impairs learning and memory abilities by inhibiting excitatory projection neuro-circuit of the hippocampus in mice

Chronic Pb exposure impairs learning and memory abilities by inhibiting excitatory projection neuro-circuit of the hippocampus in mice | 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 Chronic Pb exposure impairs learning and memory abilities by inhibiting excitatory projection neuro-circuit of the hippocampus in mice Rong-Xin Zou, Xiaozhen Gu, Chengqing Huang, Hui-Li Wang, Xiang-tao Chen This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3061407/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 Lead (Pb) is an environmental neurotoxic metal. Chronic Pb exposure causes behavioral changes in humans and rodents, such as dysfunctional learning and memory. Nevertheless, it is not clear whether Pb exposure disrupts the neural circuit. Thus, here we aim at investigating the effects the chronic Pb exposure on neural-behavioral and neural circuits in mice from prenatal to postnatal day (PND) 63. Pregnant mice and their male offspring were treated with Pb (150 ppm) until postnatal day 63. In this study, several behavior tests and Golgi-Cox staining methods were used to assess spatial memory ability and synaptogenesis. Virus-based tracing systems and immunohistochemistry assays were used to test the relevance of chronic Pb exposure with disrupted neural circuits. The behavioral experiments and Golgi-Cox staining results showed that Pb exposure impaired spatial memory and spine density in mice. The virus tracing results revealed that Entorhinal cortex (EC) neurons could be directly projected to CA1 and DG, forming a critical circuit inhibited, in either a direct or indirect way, by Pb invasion. In addition, excitatory neural input from EC(labeled with CaMK2)to CA1/DG was significantly attenuated by Pb exposure. In conclusion, our data indicated that Pb significantly impaired the excitatory connections from EC to the hippocampus (CA1 and DG), providing a novel neuro-circuitry basis for Pb neurotoxicity. Lead (Pb) neural circuit learning and memory virus tracing Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 1. Introduction Heavy metal pollution is a widespread environmental issue. Pb is recognized as one of the pervasive environmental toxicants (Neal and Guilarte, 2010 ; Amos-Kroohs et al., 2016 ). The toxic effects of Pb, especially for children, have become a public health problem (Luo et al., 2012 ; 2013; Grandjean and Landrigan, 2014 ). Pb exposure can cause cognitive impairment and inattention (Canfield et al., 2003 ; Lidsky and Schneider, 2003 ; 2005). Developmental Pb exposure has been considered a high-risk factor for attention deficit hyperactivity disorder (ADHD) in children (Wang et al., 2008a ). During the development period, Pb inhibits hippocampal synaptic transmission (Ding et al., 2018 ) and causes dendritic deficits in hippocampal pyramidal neurons of SD rats (Hu et al., 2014 ). The mechanism of Pb-induced neurotoxicity is known to disrupt neurotransmitter release (Neal and Guilarte, 2010 ), affecting the expression and function of neurotransmitter receptors in vivo and in vitro experimental models (Wang et al., 2008b ; Sanders et al., 2009 ). Hippocampal dysfunction was widely viewed as a key contributory step of Pb-mediated cognitive deficits in rats (Wang et al., 2016 ; Ding et al., 2018 ). The hippocampus, a key brain region of mammalian, forms a constituent of the limbic system which belongs to the forebrain and encodes learning and memory, stress, and anxiety regulation (Leuner and Gould, 2010 ). A prior study indicated anatomical segregation of hippocampal Function in rodents (Fanselow and Dong, 2010 ). Spatial memory appears to rely on the dorsal hippocampus (DH), not the ventral hippocampus(VH), to regulate emotional behavior and stress responses (Moser et al., 1995 ). VH, but not DH, regulates emotional behavior and stress responses. In our previous study, Pb exposure has been found to mediate spatial learning and memory impairment in SD rats. Meanwhile, a concomitant reduction of spine density was observed in hippocampal CA1 and DG areas in vivo (Zhang et al., 2013 ) and in vitro(Hu et al., 2014 ; Yang et al., 2016a; Wu et al., 2018 ). It also inhibited the excitatory transmission in primary cultured hippocampal neurons and acute hippocampal slices (Ding et al., 2018 ). To date, no involvement of hippocampus-related neural circuits was identified in Pb-induced neurobehavioral dysfunction. On the other hand, the hippocampus is a medial temporal lobe structure that is critically involved in spatial navigation and the formation of declarative memory. Multimodal information from many cortices converges into the hippocampus primarily through the EC. EC conveys spatial and content-related information of mice into the hippocampus, through two synaptic pathways: the perforant path (PP), from EC to hippocampal DG, and the temporoammonic path (TA), from EC to hippocampal CA1 (Li et al., 2017 ). Overall, the neural network of the EC→hippocampus plays an important role in modulating memories of varying subtypes. Based on its importance, it’s hypothesized that the deregulation of dorsal hippocampus neural circuit might be implicated in cognitive disorders with environmental etiology. To test this hypothesis, we performed several behavioral experiments to assess the impact of Pb on hippocampus-dependent memory and cognition in Thy1-Cre mice. Using virus-based tracing approaches, we examined the input projection of CA1 and DG pyramidal cell alterations after Pb exposure, characterized by the aberrant ascending pathway of the hippocampus. This finding might first shed light on the neuro-circuit mechanisms of Pb neurotoxicity. 2. Materials and Method Experimental animals and treatment C57BL/6 mice were supplied by the Laboratory Animal Center, Anhui Medical University, P.R. China. Thy1-Cre mice (Jackson Laboratory stock number:006143, a kind gift from the lab of Fuqiang Xu, Wuhan Institute of Physics and Mathematics (WIPM) of Chinese Academy of Sciences) is a GPI-linked glycoprotein expressed in developing nervous tissue. As a transgenic recombinase-expressing animal, cre recombinase expression was driven in pyramidal neurons in Thy1-Cre mice forebrain regions including the hippocampus (Sugino et al., 2006 ). Therefore, we were able to explore the excitatory neural projection for the hippocampus with Thy1-labelled glutamatergic pyramidal neurons (Jasnow et al., 2013 ). Thy1-Cre and C57BL/6 mice were fed with laboratory chow and distilled water and individually housed in an ambient temperature (20 ± 2°C) and relative humidity (50 ± 10%) controlled environment on a 12 h-12 h light-dark cycle. All the animals were crossed by C57BL/6 and Thy1-Cre mice. The protocol of Pb exposure in vivo was carried out as described previously(Wu et al., 2018 ). Three-month-old female (20 ± 2 g) Thy1-Cre mice (n = 12) were kept for a week in a cage with three-month sexually C57BL/6 mature males (25 ± 2 g, 2:1). After a week, they were separated from the males, and each female was placed in an individual cage. Pregnant females were divided into two groups: control and Pb-exposed, each dam could give birth to 2–5 Thy1-Cre male offspring on average. Females from the Pb- exposed group (n = 3) received 150ppm Pb acetate (PbAc) in drinking water ad libitum, starting from the first day of gestation. Pregnant females from the control group received drinking water until weaning of the offspring. During the feeding of pups, mothers from the Pb- exposed group were still receiving PbAc in drinking water ad libitum. Pups were weaned at postnatal day 21 (PND 21) and placed in separate cages. From that moment, young mice in both groups received drinking water or 150 ppm PbAc in drinking water until PND 63, respectively. All mice used in the experiment were offspring adult (9–13 weeks) male mice with a weight of 22 ± 2 g. When 9–13 weeks old, part of the male Thy1-Cre mice (N control =10, N Pb =10) was subjected to Barnes /MWM/Fear-memory tests in order, and other male Thy1-Cre mice(n = 6 per group) were subjected to virus injection. The behavioral experiments were conducted in this order because the Barnes maze, the water maze, and the Fear-memory test experiments caused stressful responses in mice in increasing order. All animal procedures were carried out following the National Institute of Health Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee of Hefei University of Technology, China. Figure 1 illustrates the research design timeline. Morris water maze (MWM) experiments The protocol of the MWM test in mice was similar to the previous study (da Silva et al., 2018 ). The MWM consisted of a blue circular pool filled with water at 24–27°C (diameter 100 cm, height 35 cm), situated in a quiet room with an ambient temperature (20 ± 2°C) controlled environment. Enough milk was added to the water to make it opaque and to assure that the escape platform would be hidden. The tank is divided into four equal quadrants with four different geometric figures (red square, yellow circle, blue triangle, and green pentagram respectively) marked on the wall of the tank as visual cues. The geometric figures were hung on the inner wall of the tank every 90°, about 15–20 cm above the water surface. Moreover, the test room presented many visual references either endogenous (i.e.: door, the recording system, and the researcher). All the references (including the researcher) stood in the same place throughout all training and test days. Mice were trained to find a platform hidden 1.5 cm below the water surface, at a fixed location in one of the maze’s quadrants. Mice were given 4 trials/day for 5 consecutive days to find the hidden platform. After the last trial, the mice were placed in the pool without the platform for 60 s for the probe test. Data were collected using a computerized video-tracking system (Anymaze software, Shanghai Xinruan Information Technology Co. Ltd, China). Barnes maze In the Barnes maze, the animals are trained to use spatial clues mice to find a small dark escape chamber under the platform called the “escape box.” The maze consists of a white circular platform, 1 m in diameter with 20 evenly spaced holes at the edges. The platform is elevated 1 m from the ground to prevent animals from jumping off. The escape tunnel is retained at the same position relative to the room, while the platform is rotated with each trial to prevent any possible scent trails. The test room presented some visual references (i.e.: the door, geometric figures marked on the wall, and the researcher). All references stood in the same place throughout all training and test days. Mice were individually placed in the escape box from the target hole to adapt for 3 min one day before the test. Place the mouse in a plastic drum (20cm in diameter and 27cm in height) in the center of the maze and limit its activities to 5s. Remove the drum, start the timer and the experimenter observes behind the curtain. If the animal’s limbs enter the target box, it is counted as an escape, and the animal is allowed to stay in the box for 30s. Each animal was observed for 3 min. During this period, if the animal still cannot find the target box, remove the animal from the maze, put it in the target box, and stay for 30s. Animals are trained twice a day for 6 days. Starting from the second training, the maze is randomly rotated one to several holes before each training, but the target box is always fixed in the same position. The purpose of this is to prevent animals from relying on smell, rather than memory to determine the location of the target hole. On the 6th day after the end of the training, the test was carried out without an escape box. At each training interval, we remove the used bedding and wipe the escape box with 70% alcohol to eliminate the interference of odors. Fear Conditioning The protocol of fear conditioning was carried out as described previously (Shoji et al., 2014 ; Chew et al., 2015 ). The mice were acclimated to the training room and handled for 2 days before training. Mice were placed in individual chambers for 2 min, after which a white noise tone (conditioned stimulus (CS)) was sounded for 30 s. The sound was co-terminated with a 0.6-mA footshock (unconditioned stimulus (US)) for 2 sec during the last 2 sec of the sound. The mice received three CS-US pairings in an 8 min session. The mice were immediately removed and placed back into their home cages. Each chamber was cleaned. To test for context-dependent fear conditioning, the mice were placed back in the same boxes 24 h later without tone or shock, and their behavior was videotaped for 5 min. The amount of time the animal spent freezing was assessed by the Freeze Frame program (Actimetrics). Freezing behavior was defined as no movement except for respiration. The cued test is performed 2 h after the context test for 6 min. In this test, mice are placed into another testing chamber that has a different shape, providing a new context that is unrelated to the conditioning chamber for the first 3 min, then the white noise is presented at the end of the last 3 min. Genotyping To examine the mouse genotype we extracted DNA from the edge of the tail tissue using a DNeasy tissue kit (TransGen Biotech). The genes of interest were amplified PCR (Primers: F: 5’-GCGGTCTGGCAGTAAAAACTATC. R: 5’-GTGAAACAGCATTGCTGTCACTT) and the samples were run on an agarose gel (1% agarose in Tris buffer) using ethidium bromide as a DNA florescent indicator. Lead concentration determination For the Pb concentration assay, the tissue sample of the hippocampus (0.03–0.05 g) was added with nitric acid (excellent pure GR, 4 mL) and 30% hydrogen peroxide (AR, 2 mL) in a nitrolysis tube overnight at room temperature, then hydrolyzed for 30 min in the microwave nitrate pyrolysis furnace (MARSXpress, CEM Corporation, USA). Lastly, the Pb concentration within the sample (without any solids) was detected by the graphite furnace atomic spectrophotometry (The PerkinElmer AAnalyst 800, USA). Blood Pb level assay was performed as follows: The mice were executed with CO 2 and fresh blood was immediately collected through the celiac artery in an anticoagulant tube containing anticoagulant (sodium heparin) to mix the blood with the anticoagulant. 0.5 mL of blood was added into 4.5 mL dilution liquid (0.2% nitric acid and 0.1% TritonX-100). Each sample was vortexed for 2 min and the Pb level within the sample was detected by the graphite furnace atomic spectrophotometry (The PerkinElmer AAnalystTM800, USA). Injection of virus The virus injection experiment started with 2-month-old Thy1-Cre mice. Briefly, animals were anesthetized with Pentobarbital sodium (40mg/kg, i.p.), and then placed in a stereotaxic apparatus (RWD, 68030). Firstly, the mixture of Cre-dependent helper viruses (AAV-EF1a-DIO-GT and AAV-EF1a-DIO-RV-G, 1:1; green) was injected into CA1 (AP:-1.7mm, ML:-1.05mm, DV:-1.48mm; 80nL) and DG (AP:-1.7mm, ML:-1.0mm, DV:-2.00mm; 100nL) of Thy1-Cre mice. After a month of full expression, EnvA-pseudotyped RV-DG-DsRed (red; mutant rabies virus with glycoprotein G gene deletion) was injected at the same coordinates to retrogradely track upstream areas of CA1 and DG. The helper virus allows the rabies virus spread one synapse retrogradely. Mice were killed 1 week after rabies infection. AAV and RV were purchased from the BrainVTA Company. All procedures on animals were performed in Biosafety level 2 (BSL2) animal facilities. Golgi-Cox staining and spine density assay The brain was processed by the Golgi-Cox staining method, which is a well-known method used to stain whole neuron dendrites and spines in vivo (Gibb and Kolb, 1998 ; Koyama and Tohyama, 2013 ). The Golgi-Cox staining method was performed as described previously (Yang et al., 2016a; Wu et al., 2018 ). In brief, the brains were removed and stored in a dark place (37℃) for two days in Golgi-Cox solution. Then the brains were sectioned at a thickness of 200 mm in the coronal plane with a vibratome (VT1000S, Leica, Germany). All sections were collected on 2% gelatin-coated slides. Then slices were stained with ammonia for 60 mins, washed with water 3 times, followed by Kodak Film Fix for 30 mins, and then washed with water, dehydrated, cleared, and mounted using a resinous medium. Approximately 70 granule or pyramidal cells randomly chosen from the intact cells occurring in a microscopic field were imaged with a wide field microscope (Eclipse 80i, Nikon) using a 40x objective. From all sections in each mouse, about 10 neurons per brain of animals were chosen. Then spine densities were calculated as mean numbers of spines per 10µm per dendrite per neuron in individual mice per group by using Image J. The spines in the 2, and 3 stretches of the secondary dendrite were counted. Immunohistochemistry Mice were deeply anesthetized and perfused transcardially with PBS, followed by ice-cold 4% paraformaldehyde. Brains were removed carefully and post-fixed in PBS containing 4% paraformaldehyde at 4℃ overnight and cryoprotected in 30% sucrose solution for 3 days. Coronal brain slices (40µm thick) were sectioned and the antibodies rabbit anti-CamKII (Abcam) were used as follows. After washing with PBS, the sections were incubated in the secondary antiserum anti-rabbit IgG (dilution 1:100, 711-607-003, Jackson). Fluorescence images of virus tracing neurons were captured with a Leica TCS SP1 Confocal microscope by using a 60×oil and 100×oil objective. Olympus OlyVIA and Leica LAS X software were used for image acquisition and analysis. For cell counting, the boundaries of brain regions were delineated manually with Photoshop based on the Allen Brain Atlas. The labeled neurons were quantified semi-automatically using FIJI and the cell counter plugin of ImageJ. Data analyses All data were expressed as Mean ± S.E.M. Statistical analysis was performed using the GraphPad Prism software (version 6.0). Initially, all the data were subjected to the Shapiro-Wilknormality test. Original data were transformed for further parametric analysis when they did not present normal distribution. Next, they were subjected to an analysis of variance according. The statistical significance of differences among groups were performed using an unpaired t-test. In the case of a non-parametric distribution of data, Mann–Whitney U test was used. Asterisk symbols on columns indicate the statistical significance between the groups. *, ** and *** represent P < 0.05, P < 0.01 and P < 0.001, respectively. 3. Results 3.1 Pb exposure impairs spatial memory and contextual fear memory of Thy1-cre mice Morris water maze and Barnes maze were adopted to test the ability of spatial learning and memory in Thy1-Cre mice. As seen in Fig. 2 below, Pb exposure significantly impaired spatial memory in Thy1-Cre mice. Duration training (four times a day and 10 min intervals) days, the Pb-exposed animals displayed a reduction in average latency, in comparison with controlled mice (Fig. 2 A). Probe tests showed that Pb exposure decreased the duration spent in the target quadrant (P = 0.0003, t = 4.528, unpaired t-test, Fig. 2 C) and the number of crossing platform (P = 0.012, U = 13.0, Mann-Whitney test, Fig. 2 D) without affecting the locomotion of the mice (Fig. 2 B). Because the MWM test represents an artificial situation and is relatively stressful for mice, a less stressful and ethologically more relevant spatial-memory assay, the Barnes maze was conducted to study hippocampus-dependent spatial learning. As the training days increased, it was shown that Pb-exposed mice need more time to get the first entry compared with Control mice (Fig. 2 E). Probe tests showed Pb exposure Pb to longer latency to find the first entry (P = 0.004, U = 7.5, Mann-Whitney test, Fig. 2 F) and more errors to enter the wrong entry (P = 0.0005, t = 4.369, unpaired t-test, Fig. 2 G). In addition, the time spent in the target room was also decreased. These observations revealed severe toxic concerns in Pb-induced spatial memory impairment. In addition to spatial memory, the hippocampus is also essential for consolidating contextual memory. Fear conditioning tasks offer a test of contextual memory where performance is motivated by emotion. The test can assess the ability of mice to learn and remember an association between environmental cues and aversive experiences. To assess if Pb exposure affects contextual memory, we examined mice using a contextual fear-conditioning task (Fig. 2 H). During the habituation phase of training, both groups showed no performance of freezing. Subsequently, following three CS-US pairings shock training, both groups showed significantly increased freezing which indicated that contextual fear memory was successfully acquired at this stage (Fig. 2 I). After an additional 24h of fear conditioning, both groups were assessed for context test and cued test. As revealed by the results, the Pb-exposed exposure group showed significantly lower freezing times as compared to the control (P = 0.0017, t = 3.822, unpaired t-test, Fig. 2 J) in the context test, while Pb exposure did not significantly affect the cued fear memory in a significant way (P = 0.663, t = 0.444, unpaired t-test). It indicated Pb exposure produces a deficit in spatial (context fear) but not nonspatial (cued fear) memory. 3.2 Pb exposure decreases spine density of Thy1-cre mice To investigate why chronic Pb exposure affects learning and memory, blood and brain tissue Pb levels in mice were examined. The results showed that both blood (P = 0.004, U = 0, Mann-Whitney test, Fig. 3 A) and brain tissue Pb levels (P = 0.002, U = 0, Mann-Whitney test, Fig. 3 B) were significantly increased in the Pb-exposed group compared with the control group. The hippocampus is an important brain region responsible for learning and memory. Dendritic spines on neurons in the hippocampus are the main structural foundation of excitatory synapses and the basic structure of neural circuit connections. Changes in dendritic spine density are crucial for post-synaptic plasticity and contribute to the morphological bases of learning and memory function (Nimchinsky et al., 2002 ). The morphology of the neurons in the CA1 and DG region of hippocampi of mice was shown in representative graphs of Fig. 3 C. Golgi-Cox staining results showed Pb exposure significantly decreased the spine density both in CA1 (P < 0.0001, t = 19.03, unpaired t-test, Fig. 3 D) and DG (P < 0.0001, U = 97.5, Mann-Whitney test, Fig. 3 E). 3.3 Tracing the upstream regions of CA1 and DG in Pb exposure Thy1-Cre mice Since DH and VH account for spatial memory and emotional behavior, respectively (Moser et al., 1995 ), Pb exposure is assumed to affect DH instead of VH, based on the aforementioned findings. We attempt to trace the upstream regions of DH in Pb-exposed Thy1-Cre mice. Firstly, AAV helper (green) was injected into dorsal CA1 (Fig. 4 A) and DG (Fig. 4 C), respectively. After a month, RV (red) was injected into the same site. The double labeling neurons (yellow) showed co-expression of AAV and RV (Fig. 4 B and D), which were then counted as starting neurons to track upstream regions of CA1 and DG regions, respectively. The results showed no significant difference in the number of AAV/RV-labeled starting neurons between the two groups in CA1 (P = 0.745, t = 0.335, Fig. 4 E) and DG (P = 0.478, t = 0.737, Fig. 4 F) injection sites respectively, indicating that there was no significant difference in the labeling efficiency of AAV between the two groups. 3.4 Pb exposure reduced projections of CA3→CA1 and EC→CA1 Firstly, we confirmed that CA1 directly received projection from CA3 in interior hippocampus and EC region. Then, we estimated effect of Pb on CA3→CA1 and EC→CA1 circuit with cell counts. The number of starting neurons shows no difference in control and Pb groups. We found the inputs ratio of RV + neurons in CA3 projecting to CA1 starting neurons (P = 0.029, t = 2.537, unpaired t test, Fig. 5 A, B) was significantly decreased. And the similar situation also arose from pairwise comparisons of EC neurons projecting to CA1 (P = 0.0228, t = 2.687, unpaired t test, Fig. 5 C, D). This is a direct proof that the neural circuit of CA3→CA1 and EC→CA1 was damaged due to the exposure of Pb in the studied dosage, which might be implicated in the Pb-mediated injury of hippocampus dependent memories. 3.5 Pb exposure reduced projections of EC→DG We confirmed that DG also receives projections from EC. Then, we estimated the effect of Pb on the EC→DG circuit with inputs ratio. Results showed Pb treatment could lead to a prominent reduction of projection from EC to DG (P = 0.044, t = 2.301, unpaired t-test, Fig. 6 ), suggesting that the EC→DG pathway was also impaired due to Pb toxicity. . 3.6 The projection of CA3 CaMK2 + neurons and EC CaMK2 + neurons to CA1 projective neurons was inhibited by Pb exposure respectively As a variety of cells co-existed in a prespecified brain region, it was essential to investigate which type of neuronal projection was affected by Pb exposure. In the following experiment, CaMK2 + was used to stain the excitatory neurons in CA3 and EC (Fig. 7 A). It’s shown from the co-localization of CaMK2 + and RV + neurons, the excitatory inputs of CA1 from CA3(P = 0.002, t = 4.030, unpaired t-test, Fig. 8 A) and EC(P = 0.002, t = 4.114, unpaired t-test, Fig. 7 B) was significantly reduced after Pb exposure, which revealed Pb exposure inhibited the excitatory neuronal input of CA3/EC to CA1. 3.7 The projection of EC CaMK2 + neurons to DG projective neurons was inhibited by Pb exposure CaMK2 was used to mark the excitatory neurons in EC. The result showed Pb exposure decreases the excitatory inputs from EC to DG (P = 0.009, U = 2.0, Mann-Whitney test, Fig. 8 ), according to calculating the proportion of CaMK2 + cells in RV + cells. In summary, these data suggested that Pb was very likely to impair the learning and memory abilities of Thy1-Cre mice by inhibiting excitatory projection from upstream regions to the hippocampus. Discussion The effects of Pb exposure on the nervous system manifested in different levels and dimensions (White et al., 2007 ; Santa Maria et al., 2018 ). Firstly, as a heavy metal poison from environment and food, Pb can interact with genes and affect the expression of the immediate early gene (IEG), which is closely related to physiological processes such as neuronal growth, differentiation, information transfer, learning, and memory after entering the organism. Pb can induce the overexpression of the early genes c-fos and c-jun in brain tissue, which further causes damage to the nervous system. Heavy metal Pb also causes a series of abnormalities in epigenetic regulation (Schneider JS, 2013), leading to alterations in DNA methylation and histone modifications, through early inhibition of histone methyltransferase EZH2 to decrease the expression of histone H3K27me3, resulting in a decrease in the density of dendritic spines in the hippocampus(Gu XZ, 2019). It has been shown that Pb weakens the wnt pathway by inhibiting the expression of Wnt7a and thus decreases dendritic spine density in hippocampal neurons, which may be a potential target for Pb-induced CNS damage during synaptogenesis. The effects of Pb exposure on individual behavior are mainly manifested in the effects on learning and memory capacity and intelligence, causing a decline in organic memory and cognitive abilities(Gu XZ, 2019), For developing children, the immature blood-brain barrier makes them more susceptible to Pb toxicity. Besides, in the World Health Organization report on Pb and environmental assessment, blood Pb levels in infants, toddlers, and preschoolers are significantly negatively correlated with intelligence quotient (IQ) values, and the average IQ of children with blood Pb levels of 50–99 µg/L is 4.9 points smaller than that of children with blood Pb levels less than 50 µg/L(Jusko et al., 2008 ). In recent years, many studies have shown that long-term and low-dose Pb exposure during development may be an important risk factor for neurodegenerative diseases such as Alzheimer's disease (Bihaqi SW, 2013 ; Fenga C, 2016; Chin-Chan M, 2019). In-depth studies on the mechanisms of neurological damage from Pb exposure are not only of scientific significance but also of social importance. Previous work in our lab has demonstrated that chronic Pb exposure impairs spatial memory through the wnt signaling pathway in SD male rats (Hu et al., 2014 ; Wu et al., 2018 ). Nonetheless, there are still some major questions remaining to be clarified. Whether and how Pb exposure alters the network and neuronal connection of the hippocampus, for instance, is one of the unresolved issues which started to be unveiled in this study. To address these questions, we performed MWM, Barnes maze, and contextual fear-conditioning tests in Thy1-Cre mice and discovered that Pb exposure induced the impairment of spatial memory and contextual fear memory. In particular, using a virus tracing system, we found that Pb exposure inhibited the upstream projections to CA1 (CA3→CA1/EC→CA1) and DG (EC→DG). Moreover, immunohistochemistry experiments showed that excitatory projections of CA3→CA1 / EC→CA1 and EC→DG were negatively affected by Pb exposure. Memory formation, consolidation, and retrieval require efficient and functional neuronal networks. Hippocampus is a medial temporal lobe structure that is critically involved in spatial navigation and the formation of declarative memory. Multimodal information from many cortices converges into the hippocampus primarily through the EC (van Strien et al., 2009 ; Buzsaki and Moser, 2013 ). EC transfer spatial and nonspatial (Hargreaves et al., 2005 ; Zhang et al., 2013 ), or content-related (Knierim et al., 2014 ) information into the hippocampus, through two synaptic pathways: the perforant path (PP), from EC to hippocampal DG, and the TA, from EC to hippocampal CA1 (Li et al., 2017 ). There is a circuit in the hippocampus with input from the EC that forms connections with the DG and output back to the EC through CA1. DG also plays an important role in learning and memory by processing and representing spatial information based on conjunctive encoding, pattern separation, and encoding of spatial information in conjunction with the CA3 (Kesner, 2007 ). It receives multiple sensory inputs including vestibular, olfactory, visual, auditory, and somatosensory from the perirhinal cortex and lateral entorhinal cortex in conjunction with spatially organized grid cells from the medial entorhinal cortex (Hafting et al., 2005 ). Damage to any part of this circuit will affect the process of learning and memory (Conrad and Roy, 1995 ; Kadar et al., 1998 ; Ogura et al., 2002 ; Lee et al., 2005 ). While increasing evidence implicated the cellular, subcellular, and molecular aberrations in Pb neurotoxicity, the alterations of neural circuits were not fully underscored. This attempt provides novel empirical evidence to encompass EC→DG/EC→CA1 disruptions into serious considerations of memory loss caused by Pb. Thus, the neuro-network structure, beyond the span of normal activity, also mediated the adverse memory specifications. Still, it warrants future inspections if the reduced neural circuits resulted from the loss of EC neurons or the weakened inter-regional connections. For several decades, the tri-synaptic circuit EC→DG→CA3→CA1 has been considered the primary substrate for learning and memory (Kohara et al., 2014 ). In the hippocampus, Long-term potentiation (LTP) is considered the basis of learning and memory. The synaptic transmission of CA3→CA1 through the Schaffer collateral pathway is conventionally NMDAR-dependent (Bliss and Collingridge, 1993 ). Our previous studies indicated that Pb exposure induced spine density deficits of CA1 and DG in SD rats and an imbalance of excitatory and inhibitory synaptic transmission in cultured rat hippocampal neurons (Wu et al., 2018 ; Zou et al., 2020 ). Thus we reasonably hypothesize that the disorder of learning and memory abilities is related to neural circuits within the EC →hippocampus network. Some experimental evidence has revealed the perforant path (EC→DG→CA3→CA1) and its critical roles in spatial navigation and declarative memory formation (Yassa and Stark, 2011 ; Basu and Siegelbaum, 2015 ), the direct paths (EC→CA1) also play important roles in spatial memory. In particular, the DG of the hippocampus is also critical to the encoding of contextual fear memories (Rao-Ruiz et al., 2019 ). EC→DG→CA3 pathway is crucial for forming discriminatory representations of similar spaces or contexts (Kitamura et al., 2015 ). These exhibited a prespecified memory-related neuro-circuitry available for potential exotic Pb interference. Our data showed that Pb exposure not just inhibited the perforant path (EC→DG→CA3→CA1) from excitatory EC neurons to DG, but also impaired the direct paths (excitatory EC neurons→CA1). As a vital circuit, EC→CA1 was also closely associated with learning and memory (Li et al., 2017 ). Some research indicated that excitatory projection played an important role in associational memory from EC to pyramidal neurons in CA1 (Yang et al., 2016b ). In addition, the preceding results identify a direct EC→CA1 circuit that is required for olfactory associative learning (Li et al., 2017 ). Thus, the adverse consequences of Pb were robust and represented by multiple circuit dysregulations, which was consistent with the multi-factorial properties of Pb neurotoxicology, as previously described. Conclusion In summary, the present study focused on the changes of excitatory projection directed towards CA1 and DG, in response to developmental Pb exposure. Based on our data, developmental Pb exposure impairs learning and memory abilities via inhibiting excitatory neurocircuit with relevance to the hippocampus, shedding light on the neuro-circuit mechanisms underlying Pb-induced neuronal deficits. Declarations Ethical Approval All animal procedures were carried out following the National Institute of Health Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee of Hefei University of Technology, China. Competing interests The authors declare no conflict of interest. Authors' contributions Hui-Li Wang and Xiang-tao Chen designed and supervised all the experiments and wrote the manuscript; Rong-Xin Zou performed the experiment of virus injection, Morris water maze (MWM) experiments, Barnes maze, Fear Conditioning, Genotyping; Xiaozhen Gu counted the cell number and Immunohistochemistry; Chenqing Huang performed Golgi-Cox staining and spine density assay. All authors read and approved the final manuscript. Funding This work was supported by the National Science Foundation of China (No. 82073592, 81773475, 81673624, 82274124，82273666, 82204084), the National Key Basic Research Program of China (No. 2018YFC1602201, 2018YFC1602204, 2012CB525003), the Key Laboratory of Xin’an Medicine Ministry of Education, Anhui University of Chinese Medicine (No. 2018xayx01). Availability of data and materials The data that support the findings of this study are available from the corresponding author, upon reasonable request. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3061407","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":211352746,"identity":"868c35e4-42e5-45e2-a55b-12f39b0ea4b5","order_by":0,"name":"Rong-Xin Zou","email":"","orcid":"","institution":"University of Chinese Medicine，Hefei，Anhui，230012，PR","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Rong-Xin","middleName":"","lastName":"Zou","suffix":""},{"id":211352747,"identity":"4f26a64e-3b9c-4924-86e8-29cf5e676693","order_by":1,"name":"Xiaozhen Gu","email":"","orcid":"","institution":"Hefei University of Technology","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Xiaozhen","middleName":"","lastName":"Gu","suffix":""},{"id":211352748,"identity":"a6b9814d-02fd-43ad-896f-d5aa1a9dee78","order_by":2,"name":"Chengqing Huang","email":"","orcid":"","institution":"Hefei University of Technology","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Chengqing","middleName":"","lastName":"Huang","suffix":""},{"id":211352749,"identity":"42e774fa-6384-4dec-b256-0279c2be9e02","order_by":3,"name":"Hui-Li Wang","email":"","orcid":"","institution":"Hefei University of Technology","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Hui-Li","middleName":"","lastName":"Wang","suffix":""},{"id":211352750,"identity":"0dd1b158-6eda-448e-95ea-b289d263041b","order_by":4,"name":"Xiang-tao Chen","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAwUlEQVRIiWNgGAWjYDACCTBpA+WxEa0lIQ2qmgQth0nQwj+7+dhj3h/nE+fPb37A8KHsMFCkgYAld46lG85IuJ244RibAeOMc4eBIgfwazGQyDGT+ADSwsbDwMzbdhgokkBIS/43iYSEc4nz24Ba/hKnJYcNaMuBxIZjQC2MxGiRuJFmJjkjLdl4w7E0g4M959J5JG4Q0MI/I/mZNI+Nnez85sMPH/wos5bjn0FACwo4AMQ8JKgfBaNgFIyCUYALAACpoD9dOb9BFgAAAABJRU5ErkJggg==","orcid":"","institution":"Anhui Medical University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Xiang-tao","middleName":"","lastName":"Chen","suffix":""}],"badges":[],"createdAt":"2023-06-14 06:59:46","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3061407/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3061407/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":39114862,"identity":"57f3a776-0e3a-4606-975c-60053431eb42","added_by":"auto","created_at":"2023-06-26 19:18:21","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":96247,"visible":true,"origin":"","legend":"\u003cp\u003eIllustration of the overall research design timeline.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-3061407/v1/1a2d6dc16a070b8b92255558.png"},{"id":39115010,"identity":"4ea86065-6b7f-48f7-8b8e-a5cf3ef3557d","added_by":"auto","created_at":"2023-06-26 19:26:21","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":710123,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of Pb on MWM/Barnes tests in Thy1-Cre mice. (A) Escape latency in training in MWM test; (B) Mean speed in probe tests; (C) duration in target quadrant; (D) the frequency of entering the platform; (E) The latency to the first entry of control and Pb mice in training in Barnes test; (F) latency to first entry; (G) Number of errors. Data were shown as mean ± S.E.M (there are ten mice in each group). (H)The percentage of average freezing time during training was compared between control and Pb-exposed mice; the mice in both groups showed similar freezing time percentages during training. (I) During the contextual fear memory test, Pb-exposed mice (n=10) showed decreased freezing time compared with control mice (n=10). (J) During cued fear memory test, Pb-exposed mice (n=10) showed no difference compared with control mice (n=10) (**P\u0026lt;0.01 there are about eight to ten mice in each group). *, **, *** indicate the statistical significance of differences of P\u0026lt;0.05，P\u0026lt;0.01 and P\u0026lt;0.001, respectively.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-3061407/v1/98099810cc96a2829dd9c8e0.png"},{"id":39114346,"identity":"319561f0-4701-401e-ac11-855fab15014f","added_by":"auto","created_at":"2023-06-26 19:10:21","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":437456,"visible":true,"origin":"","legend":"\u003cp\u003ePb concentration in blood/brain tissue and effects of Pb on dendritic spine density of Thy1-Cre mice. (A) Pb concentration in the mice's blood. (B) Pb concentration in the mice brains. (C)Golgi-Cox staining showed dendritic arborization. The number of dendritic spines contained within 10 µm of CA1 (D) and DG (E) was calculated. N=7 for mice and n=60-70 for neurons per group. **, *** indicate the statistical significance of differences of P\u0026lt;0.01 and P\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-3061407/v1/e9075dca56782f6f72895d04.png"},{"id":39114863,"identity":"73b6fa96-7ae4-4f63-bb77-351c1eca7a19","added_by":"auto","created_at":"2023-06-26 19:18:21","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1162880,"visible":true,"origin":"","legend":"\u003cp\u003eInjection site of virus in dorsal CA1 and DG of Thy1-Cre mice. (A) and (C)Unilateral injection site of virus (AAV and RV) in CA1/DG of Thy1-Cre mice. (B) and (D)AAV helper (green) and RV (red) were successive injected to CA1/DG. The double labeling neurons (yellow) could retrogradely track upstream areas. Scale bar, 200µm. (E) and (F) Quantization of the number of starting neurons in CA1 and DG injection sites. Data were shown as mean ± S.E.M (there are six mice in each group).\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-3061407/v1/f49f0e94cb7290df609a28ed.png"},{"id":39114347,"identity":"883d0943-5111-4757-9ee4-72c0264a6d03","added_by":"auto","created_at":"2023-06-26 19:10:21","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":418625,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of Pb exposure on CA3→CA1/EC→CA1 projection. (A) The illustrations of RV\u003csup\u003e+\u003c/sup\u003e cells in CA3. Scale bar, 200µm. (B) Illustrations of RV\u003csup\u003e+\u003c/sup\u003e cells in EC. Scale bar, 200µm. (C) The ratio of inputs from CA3 to CA1（RV\u003csup\u003e+\u003c/sup\u003e neurons in CA3/starting neurons in CA1）. (D) The ratio of inputs from EC to CA1（RV\u003csup\u003e+\u003c/sup\u003e neurons in EC/starting neurons in CA1）. Data were shown as mean ± S.E.M (there are six mice in each group). *, ** indicate the statistical significance of differences of P\u0026lt;0.05, P\u0026lt;0.01, respectively.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-3061407/v1/fa5d747db7bcf81dd5e4107f.png"},{"id":39114349,"identity":"47749221-8cd7-4d04-8a70-a1ae954ce803","added_by":"auto","created_at":"2023-06-26 19:10:21","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":618691,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of Pb exposure on EC→DG projection. (A) The illustrations of RV\u003csup\u003e+\u003c/sup\u003e cells in EC. Scale bar, 200µm. (B) The ratio of inputs from EC to DG（RV\u003csup\u003e+\u003c/sup\u003e neurons in EC/starting neurons in DG）. Data were shown as mean ± S.E.M (there are six mice in each group). *indicate the statistical significance of differences of P\u0026lt;0.05.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-3061407/v1/4beba192695fcad1d4efc9ab.png"},{"id":39114352,"identity":"b8808f69-dae8-4939-ac09-5d365beed347","added_by":"auto","created_at":"2023-06-26 19:10:21","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1375353,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of Pb on the projection of CA3 and EC excitatory neurons to CA1 projective neurons. (A) and (B) The illustrations of RV, CaMK2, and merged cells in CA3. Scale bar, 200μm， and quantization of the ratio of excitatory inputs from CA3 to CA1(CaMK2\u003csup\u003e+ \u003c/sup\u003eneurons in CA3/starting neurons in CA1). (B) The illustrations of RV, CaMK2, and merged cells in EC. Scale bar, 200μm, and quantization of the ratio of excitatory inputs from EC to CA1(CaMK2\u003csup\u003e+ \u003c/sup\u003eneurons in EC/starting neurons in CA1) ..Data were shown as mean ± S.E.M (there are six mice in each group). *, ** indicate the statistical significance of differences of P\u0026lt;0.05, P\u0026lt;0.01, respectively.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-3061407/v1/0d84094d3411ad6751eca15d.png"},{"id":39114353,"identity":"49c2839f-bc24-471a-b195-5a1cad9fa7e9","added_by":"auto","created_at":"2023-06-26 19:10:21","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":927788,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of Pb on the projection of EC excitatory neurons to DG projective neurons. (A) The illustrations of RV, CaMK2, and merged cells in EC. Scale bar, 100μm. (B) Quantization is the ratio of excitatory inputs from EC to DG (CaMK2\u003csup\u003e+\u003c/sup\u003e neurons in EC/ starting neurons in DG). Data were shown as mean ± S.E.M (there are six mice in each group). ** indicate the statistical significance of differences of P\u0026lt;0.01.\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-3061407/v1/57a1994d2c7986d6539bcfd9.png"},{"id":39947124,"identity":"9fc68d31-09b5-4429-9e6c-03ee49238cef","added_by":"auto","created_at":"2023-07-13 03:44:39","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5300665,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3061407/v1/50d1b183-7ca6-4355-a69b-1d113772970d.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Chronic Pb exposure impairs learning and memory abilities by inhibiting excitatory projection neuro-circuit of the hippocampus in mice","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eHeavy metal pollution is a widespread environmental issue. Pb is recognized as one of the pervasive environmental toxicants (Neal and Guilarte, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Amos-Kroohs et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). The toxic effects of Pb, especially for children, have become a public health problem (Luo et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; 2013; Grandjean and Landrigan, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Pb exposure can cause cognitive impairment and inattention (Canfield et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Lidsky and Schneider, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; 2005). Developmental Pb exposure has been considered a high-risk factor for attention deficit hyperactivity disorder (ADHD) in children (Wang et al., \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2008a\u003c/span\u003e). During the development period, Pb inhibits hippocampal synaptic transmission (Ding et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) and causes dendritic deficits in hippocampal pyramidal neurons of SD rats (Hu et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). The mechanism of Pb-induced neurotoxicity is known to disrupt neurotransmitter release (Neal and Guilarte, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2010\u003c/span\u003e), affecting the expression and function of neurotransmitter receptors in vivo and in vitro experimental models (Wang et al., \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2008b\u003c/span\u003e; Sanders et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2009\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eHippocampal dysfunction was widely viewed as a key contributory step of Pb-mediated cognitive deficits in rats (Wang et al., \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Ding et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). The hippocampus, a key brain region of mammalian, forms a constituent of the limbic system which belongs to the forebrain and encodes learning and memory, stress, and anxiety regulation (Leuner and Gould, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). A prior study indicated anatomical segregation of hippocampal Function in rodents (Fanselow and Dong, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Spatial memory appears to rely on the dorsal hippocampus (DH), not the ventral hippocampus(VH), to regulate emotional behavior and stress responses (Moser et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e1995\u003c/span\u003e). VH, but not DH, regulates emotional behavior and stress responses. In our previous study, Pb exposure has been found to mediate spatial learning and memory impairment in SD rats. Meanwhile, a concomitant reduction of spine density was observed in hippocampal CA1 and DG areas in vivo (Zhang et al., \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2013\u003c/span\u003e) and in vitro(Hu et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Yang et al., 2016a; Wu et al., \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). It also inhibited the excitatory transmission in primary cultured hippocampal neurons and acute hippocampal slices (Ding et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). To date, no involvement of hippocampus-related neural circuits was identified in Pb-induced neurobehavioral dysfunction. On the other hand, the hippocampus is a medial temporal lobe structure that is critically involved in spatial navigation and the formation of declarative memory. Multimodal information from many cortices converges into the hippocampus primarily through the EC. EC conveys spatial and content-related information of mice into the hippocampus, through two synaptic pathways: the perforant path (PP), from EC to hippocampal DG, and the temporoammonic path (TA), from EC to hippocampal CA1 (Li et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Overall, the neural network of the EC\u0026rarr;hippocampus plays an important role in modulating memories of varying subtypes. Based on its importance, it\u0026rsquo;s hypothesized that the deregulation of dorsal hippocampus neural circuit might be implicated in cognitive disorders with environmental etiology.\u003c/p\u003e \u003cp\u003eTo test this hypothesis, we performed several behavioral experiments to assess the impact of Pb on hippocampus-dependent memory and cognition in Thy1-Cre mice. Using virus-based tracing approaches, we examined the input projection of CA1 and DG pyramidal cell alterations after Pb exposure, characterized by the aberrant ascending pathway of the hippocampus. This finding might first shed light on the neuro-circuit mechanisms of Pb neurotoxicity.\u003c/p\u003e"},{"header":"2. Materials and Method","content":"\u003cp\u003e \u003cem\u003eExperimental animals and treatment\u003c/em\u003e \u003c/p\u003e \u003cp\u003eC57BL/6 mice were supplied by the Laboratory Animal Center, Anhui Medical University, P.R. China. Thy1-Cre mice (Jackson Laboratory stock number:006143, a kind gift from the lab of Fuqiang Xu, Wuhan Institute of Physics and Mathematics (WIPM) of Chinese Academy of Sciences) is a GPI-linked glycoprotein expressed in developing nervous tissue. As a transgenic recombinase-expressing animal, cre recombinase expression was driven in pyramidal neurons in Thy1-Cre mice forebrain regions including the hippocampus (Sugino et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). Therefore, we were able to explore the excitatory neural projection for the hippocampus with Thy1-labelled glutamatergic pyramidal neurons (Jasnow et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Thy1-Cre and C57BL/6 mice were fed with laboratory chow and distilled water and individually housed in an ambient temperature (20\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C) and relative humidity (50\u0026thinsp;\u0026plusmn;\u0026thinsp;10%) controlled environment on a 12 h-12 h light-dark cycle. All the animals were crossed by C57BL/6 and Thy1-Cre mice. The protocol of Pb exposure in vivo was carried out as described previously(Wu et al., \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Three-month-old female (20\u0026thinsp;\u0026plusmn;\u0026thinsp;2 g) Thy1-Cre mice (n\u0026thinsp;=\u0026thinsp;12) were kept for a week in a cage with three-month sexually C57BL/6 mature males (25\u0026thinsp;\u0026plusmn;\u0026thinsp;2 g, 2:1). After a week, they were separated from the males, and each female was placed in an individual cage. Pregnant females were divided into two groups: control and Pb-exposed, each dam could give birth to 2\u0026ndash;5 Thy1-Cre male offspring on average. Females from the Pb- exposed group (n\u0026thinsp;=\u0026thinsp;3) received 150ppm Pb acetate (PbAc) in drinking water ad libitum, starting from the first day of gestation. Pregnant females from the control group received drinking water until weaning of the offspring. During the feeding of pups, mothers from the Pb- exposed group were still receiving PbAc in drinking water ad libitum. Pups were weaned at postnatal day 21 (PND 21) and placed in separate cages. From that moment, young mice in both groups received drinking water or 150 ppm PbAc in drinking water until PND 63, respectively. All mice used in the experiment were offspring adult (9\u0026ndash;13 weeks) male mice with a weight of 22\u0026thinsp;\u0026plusmn;\u0026thinsp;2 g. When 9\u0026ndash;13 weeks old, part of the male Thy1-Cre mice (N\u003csub\u003econtrol\u003c/sub\u003e=10, N\u003csub\u003ePb\u003c/sub\u003e=10) was subjected to Barnes /MWM/Fear-memory tests in order, and other male Thy1-Cre mice(n\u0026thinsp;=\u0026thinsp;6 per group) were subjected to virus injection. The behavioral experiments were conducted in this order because the Barnes maze, the water maze, and the Fear-memory test experiments caused stressful responses in mice in increasing order. All animal procedures were carried out following the National Institute of Health Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee of Hefei University of Technology, China. Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e illustrates the research design timeline.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003eMorris water maze (MWM) experiments\u003c/em\u003e \u003c/p\u003e \u003cp\u003eThe protocol of the MWM test in mice was similar to the previous study (da Silva et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). The MWM consisted of a blue circular pool filled with water at 24\u0026ndash;27\u0026deg;C (diameter 100 cm, height 35 cm), situated in a quiet room with an ambient temperature (20\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C) controlled environment. Enough milk was added to the water to make it opaque and to assure that the escape platform would be hidden. The tank is divided into four equal quadrants with four different geometric figures (red square, yellow circle, blue triangle, and green pentagram respectively) marked on the wall of the tank as visual cues. The geometric figures were hung on the inner wall of the tank every 90\u0026deg;, about 15\u0026ndash;20 cm above the water surface. Moreover, the test room presented many visual references either endogenous (i.e.: door, the recording system, and the researcher). All the references (including the researcher) stood in the same place throughout all training and test days. Mice were trained to find a platform hidden 1.5 cm below the water surface, at a fixed location in one of the maze\u0026rsquo;s quadrants. Mice were given 4 trials/day for 5 consecutive days to find the hidden platform. After the last trial, the mice were placed in the pool without the platform for 60 s for the probe test. Data were collected using a computerized video-tracking system (Anymaze software, Shanghai Xinruan Information Technology Co. Ltd, China).\u003c/p\u003e \u003cp\u003e \u003cem\u003eBarnes maze\u003c/em\u003e \u003c/p\u003e \u003cp\u003eIn the Barnes maze, the animals are trained to use spatial clues mice to find a small dark escape chamber under the platform called the \u0026ldquo;escape box.\u0026rdquo; The maze consists of a white circular platform, 1 m in diameter with 20 evenly spaced holes at the edges. The platform is elevated 1 m from the ground to prevent animals from jumping off. The escape tunnel is retained at the same position relative to the room, while the platform is rotated with each trial to prevent any possible scent trails. The test room presented some visual references (i.e.: the door, geometric figures marked on the wall, and the researcher). All references stood in the same place throughout all training and test days.\u003c/p\u003e \u003cp\u003eMice were individually placed in the escape box from the target hole to adapt for 3 min one day before the test. Place the mouse in a plastic drum (20cm in diameter and 27cm in height) in the center of the maze and limit its activities to 5s. Remove the drum, start the timer and the experimenter observes behind the curtain. If the animal\u0026rsquo;s limbs enter the target box, it is counted as an escape, and the animal is allowed to stay in the box for 30s. Each animal was observed for 3 min. During this period, if the animal still cannot find the target box, remove the animal from the maze, put it in the target box, and stay for 30s. Animals are trained twice a day for 6 days. Starting from the second training, the maze is randomly rotated one to several holes before each training, but the target box is always fixed in the same position. The purpose of this is to prevent animals from relying on smell, rather than memory to determine the location of the target hole. On the 6th day after the end of the training, the test was carried out without an escape box. At each training interval, we remove the used bedding and wipe the escape box with 70% alcohol to eliminate the interference of odors.\u003c/p\u003e \u003cp\u003e \u003cem\u003eFear Conditioning\u003c/em\u003e \u003c/p\u003e \u003cp\u003eThe protocol of fear conditioning was carried out as described previously (Shoji et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Chew et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). The mice were acclimated to the training room and handled for 2 days before training. Mice were placed in individual chambers for 2 min, after which a white noise tone (conditioned stimulus (CS)) was sounded for 30 s. The sound was co-terminated with a 0.6-mA footshock (unconditioned stimulus (US)) for 2 sec during the last 2 sec of the sound. The mice received three CS-US pairings in an 8 min session. The mice were immediately removed and placed back into their home cages. Each chamber was cleaned. To test for context-dependent fear conditioning, the mice were placed back in the same boxes 24 h later without tone or shock, and their behavior was videotaped for 5 min. The amount of time the animal spent freezing was assessed by the Freeze Frame program (Actimetrics). Freezing behavior was defined as no movement except for respiration. The cued test is performed 2 h after the context test for 6 min. In this test, mice are placed into another testing chamber that has a different shape, providing a new context that is unrelated to the conditioning chamber for the first 3 min, then the white noise is presented at the end of the last 3 min.\u003c/p\u003e \u003cp\u003e \u003cem\u003eGenotyping\u003c/em\u003e \u003c/p\u003e \u003cp\u003eTo examine the mouse genotype we extracted DNA from the edge of the tail tissue using a DNeasy tissue kit (TransGen Biotech). The genes of interest were amplified PCR (Primers: F: 5\u0026rsquo;-GCGGTCTGGCAGTAAAAACTATC. R: 5\u0026rsquo;-GTGAAACAGCATTGCTGTCACTT) and the samples were run on an agarose gel (1% agarose in Tris buffer) using ethidium bromide as a DNA florescent indicator.\u003c/p\u003e \u003cp\u003e \u003cem\u003eLead concentration determination\u003c/em\u003e \u003c/p\u003e \u003cp\u003eFor the Pb concentration assay, the tissue sample of the hippocampus (0.03\u0026ndash;0.05 g) was added with nitric acid (excellent pure GR, 4 mL) and 30% hydrogen peroxide (AR, 2 mL) in a nitrolysis tube overnight at room temperature, then hydrolyzed for 30 min in the microwave nitrate pyrolysis furnace (MARSXpress, CEM Corporation, USA). Lastly, the Pb concentration within the sample (without any solids) was detected by the graphite furnace atomic spectrophotometry (The PerkinElmer AAnalyst 800, USA). Blood Pb level assay was performed as follows: The mice were executed with CO\u003csub\u003e2\u003c/sub\u003e and fresh blood was immediately collected through the celiac artery in an anticoagulant tube containing anticoagulant (sodium heparin) to mix the blood with the anticoagulant. 0.5 mL of blood was added into 4.5 mL dilution liquid (0.2% nitric acid and 0.1% TritonX-100). Each sample was vortexed for 2 min and the Pb level within the sample was detected by the graphite furnace atomic spectrophotometry (The PerkinElmer AAnalystTM800, USA).\u003c/p\u003e \u003cp\u003e \u003cem\u003eInjection of virus\u003c/em\u003e \u003c/p\u003e \u003cp\u003eThe virus injection experiment started with 2-month-old Thy1-Cre mice. Briefly, animals were anesthetized with Pentobarbital sodium (40mg/kg, i.p.), and then placed in a stereotaxic apparatus (RWD, 68030). Firstly, the mixture of Cre-dependent helper viruses (AAV-EF1a-DIO-GT and AAV-EF1a-DIO-RV-G, 1:1; green) was injected into CA1 (AP:-1.7mm, ML:-1.05mm, DV:-1.48mm; 80nL) and DG (AP:-1.7mm, ML:-1.0mm, DV:-2.00mm; 100nL) of Thy1-Cre mice. After a month of full expression, EnvA-pseudotyped RV-DG-DsRed (red; mutant rabies virus with glycoprotein G gene deletion) was injected at the same coordinates to retrogradely track upstream areas of CA1 and DG. The helper virus allows the rabies virus spread one synapse retrogradely. Mice were killed 1 week after rabies infection. AAV and RV were purchased from the BrainVTA Company. All procedures on animals were performed in Biosafety level 2 (BSL2) animal facilities.\u003c/p\u003e \u003cp\u003e \u003cem\u003eGolgi-Cox staining and spine density assay\u003c/em\u003e \u003c/p\u003e \u003cp\u003eThe brain was processed by the Golgi-Cox staining method, which is a well-known method used to stain whole neuron dendrites and spines in vivo (Gibb and Kolb, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e1998\u003c/span\u003e; Koyama and Tohyama, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). The Golgi-Cox staining method was performed as described previously (Yang et al., 2016a; Wu et al., \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). In brief, the brains were removed and stored in a dark place (37℃) for two days in Golgi-Cox solution. Then the brains were sectioned at a thickness of 200 mm in the coronal plane with a vibratome (VT1000S, Leica, Germany). All sections were collected on 2% gelatin-coated slides. Then slices were stained with ammonia for 60 mins, washed with water 3 times, followed by Kodak Film Fix for 30 mins, and then washed with water, dehydrated, cleared, and mounted using a resinous medium. Approximately 70 granule or pyramidal cells randomly chosen from the intact cells occurring in a microscopic field were imaged with a wide field microscope (Eclipse 80i, Nikon) using a 40x objective. From all sections in each mouse, about 10 neurons per brain of animals were chosen. Then spine densities were calculated as mean numbers of spines per 10\u0026micro;m per dendrite per neuron in individual mice per group by using Image J. The spines in the 2, and 3 stretches of the secondary dendrite were counted.\u003c/p\u003e \u003cp\u003e \u003cem\u003eImmunohistochemistry\u003c/em\u003e \u003c/p\u003e \u003cp\u003eMice were deeply anesthetized and perfused transcardially with PBS, followed by ice-cold 4% paraformaldehyde. Brains were removed carefully and post-fixed in PBS containing 4% paraformaldehyde at 4℃ overnight and cryoprotected in 30% sucrose solution for 3 days. Coronal brain slices (40\u0026micro;m thick) were sectioned and the antibodies rabbit anti-CamKII (Abcam) were used as follows. After washing with PBS, the sections were incubated in the secondary antiserum anti-rabbit IgG (dilution 1:100, 711-607-003, Jackson). Fluorescence images of virus tracing neurons were captured with a Leica TCS SP1 Confocal microscope by using a 60\u0026times;oil and 100\u0026times;oil objective. Olympus OlyVIA and Leica LAS X software were used for image acquisition and analysis. For cell counting, the boundaries of brain regions were delineated manually with Photoshop based on the Allen Brain Atlas. The labeled neurons were quantified semi-automatically using FIJI and the cell counter plugin of ImageJ.\u003c/p\u003e \u003cp\u003e \u003cem\u003eData analyses\u003c/em\u003e \u003c/p\u003e \u003cp\u003eAll data were expressed as Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;S.E.M. Statistical analysis was performed using the GraphPad Prism software (version 6.0). Initially, all the data were subjected to the Shapiro-Wilknormality test. Original data were transformed for further parametric analysis when they did not present normal distribution. Next, they were subjected to an analysis of variance according. The statistical significance of differences among groups were performed using an unpaired t-test. In the case of a non-parametric distribution of data, Mann\u0026ndash;Whitney U test was used. Asterisk symbols on columns indicate the statistical significance between the groups. *, ** and *** represent P\u0026thinsp;\u0026lt;\u0026thinsp;0.05, P\u0026thinsp;\u0026lt;\u0026thinsp;0.01 and P\u0026thinsp;\u0026lt;\u0026thinsp;0.001, respectively.\u003c/p\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Pb exposure impairs spatial memory and contextual fear memory of Thy1-cre mice\u003cF/h2\u003e \u003cp\u003eMorris water maze and Barnes maze were adopted to test the ability of spatial learning and memory in Thy1-Cre mice. As seen in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e below, Pb exposure significantly impaired spatial memory in Thy1-Cre mice. Duration training (four times a day and 10 min intervals) days, the Pb-exposed animals displayed a reduction in average latency, in comparison with controlled mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Probe tests showed that Pb exposure decreased the duration spent in the target quadrant (P\u0026thinsp;=\u0026thinsp;0.0003, t\u0026thinsp;=\u0026thinsp;4.528, unpaired t-test, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC) and the number of crossing platform (P\u0026thinsp;=\u0026thinsp;0.012, U\u0026thinsp;=\u0026thinsp;13.0, Mann-Whitney test, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD) without affecting the locomotion of the mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003eBecause the MWM test represents an artificial situation and is relatively stressful for mice, a less stressful and ethologically more relevant spatial-memory assay, the Barnes maze was conducted to study hippocampus-dependent spatial learning. As the training days increased, it was shown that Pb-exposed mice need more time to get the first entry compared with Control mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). Probe tests showed Pb exposure Pb to longer latency to find the first entry (P\u0026thinsp;=\u0026thinsp;0.004, U\u0026thinsp;=\u0026thinsp;7.5, Mann-Whitney test, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF) and more errors to enter the wrong entry (P\u0026thinsp;=\u0026thinsp;0.0005, t\u0026thinsp;=\u0026thinsp;4.369, unpaired t-test, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG). In addition, the time spent in the target room was also decreased. These observations revealed severe toxic concerns in Pb-induced spatial memory impairment.\u003c/p\u003e \u003cp\u003eIn addition to spatial memory, the hippocampus is also essential for consolidating contextual memory. Fear conditioning tasks offer a test of contextual memory where performance is motivated by emotion. The test can assess the ability of mice to learn and remember an association between environmental cues and aversive experiences. To assess if Pb exposure affects contextual memory, we examined mice using a contextual fear-conditioning task (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH). During the habituation phase of training, both groups showed no performance of freezing. Subsequently, following three CS-US pairings shock training, both groups showed significantly increased freezing which indicated that contextual fear memory was successfully acquired at this stage (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eI). After an additional 24h of fear conditioning, both groups were assessed for context test and cued test. As revealed by the results, the Pb-exposed exposure group showed significantly lower freezing times as compared to the control (P\u0026thinsp;=\u0026thinsp;0.0017, t\u0026thinsp;=\u0026thinsp;3.822, unpaired t-test, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eJ) in the context test, while Pb exposure did not significantly affect the cued fear memory in a significant way (P\u0026thinsp;=\u0026thinsp;0.663, t\u0026thinsp;=\u0026thinsp;0.444, unpaired t-test). It indicated Pb exposure produces a deficit in spatial (context fear) but not nonspatial (cued fear) memory.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Pb exposure decreases spine density of Thy1-cre mice\u003c/h2\u003e \u003cp\u003eTo investigate why chronic Pb exposure affects learning and memory, blood and brain tissue Pb levels in mice were examined. The results showed that both blood (P\u0026thinsp;=\u0026thinsp;0.004, U\u0026thinsp;=\u0026thinsp;0, Mann-Whitney test, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA) and brain tissue Pb levels (P\u0026thinsp;=\u0026thinsp;0.002, U\u0026thinsp;=\u0026thinsp;0, Mann-Whitney test, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB) were significantly increased in the Pb-exposed group compared with the control group. The hippocampus is an important brain region responsible for learning and memory. Dendritic spines on neurons in the hippocampus are the main structural foundation of excitatory synapses and the basic structure of neural circuit connections. Changes in dendritic spine density are crucial for post-synaptic plasticity and contribute to the morphological bases of learning and memory function (Nimchinsky et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). The morphology of the neurons in the CA1 and DG region of hippocampi of mice was shown in representative graphs of Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC. Golgi-Cox staining results showed Pb exposure significantly decreased the spine density both in CA1 (P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, t\u0026thinsp;=\u0026thinsp;19.03, unpaired t-test, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD) and DG (P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, U\u0026thinsp;=\u0026thinsp;97.5, Mann-Whitney test, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Tracing the upstream regions of CA1 and DG in Pb exposure Thy1-Cre mice\u003c/h2\u003e \u003cp\u003eSince DH and VH account for spatial memory and emotional behavior, respectively (Moser et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e1995\u003c/span\u003e), Pb exposure is assumed to affect DH instead of VH, based on the aforementioned findings. We attempt to trace the upstream regions of DH in Pb-exposed Thy1-Cre mice. Firstly, AAV helper (green) was injected into dorsal CA1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA) and DG (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC), respectively. After a month, RV (red) was injected into the same site. The double labeling neurons (yellow) showed co-expression of AAV and RV (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB and D), which were then counted as starting neurons to track upstream regions of CA1 and DG regions, respectively. The results showed no significant difference in the number of AAV/RV-labeled starting neurons between the two groups in CA1 (P\u0026thinsp;=\u0026thinsp;0.745, t\u0026thinsp;=\u0026thinsp;0.335, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE) and DG (P\u0026thinsp;=\u0026thinsp;0.478, t\u0026thinsp;=\u0026thinsp;0.737, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF) injection sites respectively, indicating that there was no significant difference in the labeling efficiency of AAV between the two groups.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Pb exposure reduced projections of CA3\u0026rarr;CA1 and EC\u0026rarr;CA1\u003c/h2\u003e \u003cp\u003eFirstly, we confirmed that CA1 directly received projection from CA3 in interior hippocampus and EC region. Then, we estimated effect of Pb on CA3\u0026rarr;CA1 and EC\u0026rarr;CA1 circuit with cell counts. The number of starting neurons shows no difference in control and Pb groups. We found the inputs ratio of RV\u003csup\u003e+\u003c/sup\u003e neurons in CA3 projecting to CA1 starting neurons (P\u0026thinsp;=\u0026thinsp;0.029, t\u0026thinsp;=\u0026thinsp;2.537, unpaired t test, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, B) was significantly decreased. And the similar situation also arose from pairwise comparisons of EC neurons projecting to CA1 (P\u0026thinsp;=\u0026thinsp;0.0228, t\u0026thinsp;=\u0026thinsp;2.687, unpaired t test, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC, D). This is a direct proof that the neural circuit of CA3\u0026rarr;CA1 and EC\u0026rarr;CA1 was damaged due to the exposure of Pb in the studied dosage, which might be implicated in the Pb-mediated injury of hippocampus dependent memories.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e3.5 Pb exposure reduced projections of EC\u0026rarr;DG\u003c/h2\u003e \u003cp\u003eWe confirmed that DG also receives projections from EC. Then, we estimated the effect of Pb on the EC\u0026rarr;DG circuit with inputs ratio. Results showed Pb treatment could lead to a prominent reduction of projection from EC to DG (P\u0026thinsp;=\u0026thinsp;0.044, t\u0026thinsp;=\u0026thinsp;2.301, unpaired t-test, Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e), suggesting that the EC\u0026rarr;DG pathway was also impaired due to Pb toxicity.\u003c/p\u003e \u003cp\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003e3.6 The projection of CA3 CaMK2\u003c/em\u003e \u003csup\u003e \u003cem\u003e+\u003c/em\u003e \u003c/sup\u003e \u003cem\u003eneurons and EC CaMK2\u003c/em\u003e\u003csup\u003e\u003cem\u003e+\u003c/em\u003e\u003c/sup\u003e \u003cem\u003eneurons to CA1 projective neurons was inhibited by Pb exposure respectively\u003c/em\u003e\u003c/p\u003e \u003cp\u003eAs a variety of cells co-existed in a prespecified brain region, it was essential to investigate which type of neuronal projection was affected by Pb exposure. In the following experiment, CaMK2\u003csup\u003e+\u003c/sup\u003e was used to stain the excitatory neurons in CA3 and EC (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). It\u0026rsquo;s shown from the co-localization of CaMK2\u003csup\u003e+\u003c/sup\u003e and RV\u003csup\u003e+\u003c/sup\u003e neurons, the excitatory inputs of CA1 from CA3(P\u0026thinsp;=\u0026thinsp;0.002, t\u0026thinsp;=\u0026thinsp;4.030, unpaired t-test, Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA) and EC(P\u0026thinsp;=\u0026thinsp;0.002, t\u0026thinsp;=\u0026thinsp;4.114, unpaired t-test, Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB) was significantly reduced after Pb exposure, which revealed Pb exposure inhibited the excitatory neuronal input of CA3/EC to CA1.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e3.7 The projection of EC CaMK2\u003csup\u003e+\u003c/sup\u003e neurons to DG projective neurons was inhibited by Pb exposure\u003c/h2\u003e \u003cp\u003eCaMK2 was used to mark the excitatory neurons in EC. The result showed Pb exposure decreases the excitatory inputs from EC to DG (P\u0026thinsp;=\u0026thinsp;0.009, U\u0026thinsp;=\u0026thinsp;2.0, Mann-Whitney test, Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e), according to calculating the proportion of CaMK2\u003csup\u003e+\u003c/sup\u003e cells in RV\u003csup\u003e+\u003c/sup\u003e cells. In summary, these data suggested that Pb was very likely to impair the learning and memory abilities of Thy1-Cre mice by inhibiting excitatory projection from upstream regions to the hippocampus.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e "},{"header":"Discussion","content":"\u003cp\u003eThe effects of Pb exposure on the nervous system manifested in different levels and dimensions (White et al., \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Santa Maria et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Firstly, as a heavy metal poison from environment and food, Pb can interact with genes and affect the expression of the immediate early gene (IEG), which is closely related to physiological processes such as neuronal growth, differentiation, information transfer, learning, and memory after entering the organism. Pb can induce the overexpression of the early genes c-fos and c-jun in brain tissue, which further causes damage to the nervous system. Heavy metal Pb also causes a series of abnormalities in epigenetic regulation (Schneider JS, 2013), leading to alterations in DNA methylation and histone modifications, through early inhibition of histone methyltransferase EZH2 to decrease the expression of histone H3K27me3, resulting in a decrease in the density of dendritic spines in the hippocampus(Gu XZ, 2019). It has been shown that Pb weakens the wnt pathway by inhibiting the expression of Wnt7a and thus decreases dendritic spine density in hippocampal neurons, which may be a potential target for Pb-induced CNS damage during synaptogenesis. The effects of Pb exposure on individual behavior are mainly manifested in the effects on learning and memory capacity and intelligence, causing a decline in organic memory and cognitive abilities(Gu XZ, 2019), For developing children, the immature blood-brain barrier makes them more susceptible to Pb toxicity. Besides, in the World Health Organization report on Pb and environmental assessment, blood Pb levels in infants, toddlers, and preschoolers are significantly negatively correlated with intelligence quotient (IQ) values, and the average IQ of children with blood Pb levels of 50\u0026ndash;99 \u0026micro;g/L is 4.9 points smaller than that of children with blood Pb levels less than 50 \u0026micro;g/L(Jusko et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). In recent years, many studies have shown that long-term and low-dose Pb exposure during development may be an important risk factor for neurodegenerative diseases such as Alzheimer's disease (Bihaqi SW, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Fenga C, 2016; Chin-Chan M, 2019). In-depth studies on the mechanisms of neurological damage from Pb exposure are not only of scientific significance but also of social importance.\u003c/p\u003e \u003cp\u003ePrevious work in our lab has demonstrated that chronic Pb exposure impairs spatial memory through the wnt signaling pathway in SD male rats (Hu et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Wu et al., \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Nonetheless, there are still some major questions remaining to be clarified. Whether and how Pb exposure alters the network and neuronal connection of the hippocampus, for instance, is one of the unresolved issues which started to be unveiled in this study. To address these questions, we performed MWM, Barnes maze, and contextual fear-conditioning tests in Thy1-Cre mice and discovered that Pb exposure induced the impairment of spatial memory and contextual fear memory. In particular, using a virus tracing system, we found that Pb exposure inhibited the upstream projections to CA1 (CA3\u0026rarr;CA1/EC\u0026rarr;CA1) and DG (EC\u0026rarr;DG). Moreover, immunohistochemistry experiments showed that excitatory projections of CA3\u0026rarr;CA1 / EC\u0026rarr;CA1 and EC\u0026rarr;DG were negatively affected by Pb exposure.\u003c/p\u003e \u003cp\u003eMemory formation, consolidation, and retrieval require efficient and functional neuronal networks. Hippocampus is a medial temporal lobe structure that is critically involved in spatial navigation and the formation of declarative memory. Multimodal information from many cortices converges into the hippocampus primarily through the EC (van Strien et al., \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Buzsaki and Moser, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). EC transfer spatial and nonspatial (Hargreaves et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Zhang et al., \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2013\u003c/span\u003e), or content-related (Knierim et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) information into the hippocampus, through two synaptic pathways: the perforant path (PP), from EC to hippocampal DG, and the TA, from EC to hippocampal CA1 (Li et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). There is a circuit in the hippocampus with input from the EC that forms connections with the DG and output back to the EC through CA1. DG also plays an important role in learning and memory by processing and representing spatial information based on conjunctive encoding, pattern separation, and encoding of spatial information in conjunction with the CA3 (Kesner, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). It receives multiple sensory inputs including vestibular, olfactory, visual, auditory, and somatosensory from the perirhinal cortex and lateral entorhinal cortex in conjunction with spatially organized grid cells from the medial entorhinal cortex (Hafting et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). Damage to any part of this circuit will affect the process of learning and memory (Conrad and Roy, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e1995\u003c/span\u003e; Kadar et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e1998\u003c/span\u003e; Ogura et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Lee et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). While increasing evidence implicated the cellular, subcellular, and molecular aberrations in Pb neurotoxicity, the alterations of neural circuits were not fully underscored. This attempt provides novel empirical evidence to encompass EC\u0026rarr;DG/EC\u0026rarr;CA1 disruptions into serious considerations of memory loss caused by Pb. Thus, the neuro-network structure, beyond the span of normal activity, also mediated the adverse memory specifications. Still, it warrants future inspections if the reduced neural circuits resulted from the loss of EC neurons or the weakened inter-regional connections.\u003c/p\u003e \u003cp\u003eFor several decades, the tri-synaptic circuit EC\u0026rarr;DG\u0026rarr;CA3\u0026rarr;CA1 has been considered the primary substrate for learning and memory (Kohara et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). In the hippocampus, Long-term potentiation (LTP) is considered the basis of learning and memory. The synaptic transmission of CA3\u0026rarr;CA1 through the Schaffer collateral pathway is conventionally NMDAR-dependent (Bliss and Collingridge, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e1993\u003c/span\u003e). Our previous studies indicated that Pb exposure induced spine density deficits of CA1 and DG in SD rats and an imbalance of excitatory and inhibitory synaptic transmission in cultured rat hippocampal neurons (Wu et al., \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Zou et al., \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Thus we reasonably hypothesize that the disorder of learning and memory abilities is related to neural circuits within the EC \u0026rarr;hippocampus network. Some experimental evidence has revealed the perforant path (EC\u0026rarr;DG\u0026rarr;CA3\u0026rarr;CA1) and its critical roles in spatial navigation and declarative memory formation (Yassa and Stark, \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Basu and Siegelbaum, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), the direct paths (EC\u0026rarr;CA1) also play important roles in spatial memory. In particular, the DG of the hippocampus is also critical to the encoding of contextual fear memories (Rao-Ruiz et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). EC\u0026rarr;DG\u0026rarr;CA3 pathway is crucial for forming discriminatory representations of similar spaces or contexts (Kitamura et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). These exhibited a prespecified memory-related neuro-circuitry available for potential exotic Pb interference.\u003c/p\u003e \u003cp\u003eOur data showed that Pb exposure not just inhibited the perforant path (EC\u0026rarr;DG\u0026rarr;CA3\u0026rarr;CA1) from excitatory EC neurons to DG, but also impaired the direct paths (excitatory EC neurons\u0026rarr;CA1). As a vital circuit, EC\u0026rarr;CA1 was also closely associated with learning and memory (Li et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Some research indicated that excitatory projection played an important role in associational memory from EC to pyramidal neurons in CA1 (Yang et al., \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2016b\u003c/span\u003e). In addition, the preceding results identify a direct EC\u0026rarr;CA1 circuit that is required for olfactory associative learning (Li et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Thus, the adverse consequences of Pb were robust and represented by multiple circuit dysregulations, which was consistent with the multi-factorial properties of Pb neurotoxicology, as previously described.\u003c/p\u003e "},{"header":"Conclusion","content":"\u003cp\u003eIn summary, the present study focused on the changes of excitatory projection directed towards CA1 and DG, in response to developmental Pb exposure. Based on our data, developmental Pb exposure impairs learning and memory abilities via inhibiting excitatory neurocircuit with relevance to the hippocampus, shedding light on the neuro-circuit mechanisms underlying Pb-induced neuronal deficits.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthical Approval\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll animal procedures were carried out following the National Institute of Health Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee of Hefei University of Technology, China.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors' contributions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHui-Li Wang and Xiang-tao Chen designed and supervised all the experiments and wrote the manuscript;\u0026nbsp;Rong-Xin Zou\u0026nbsp;performed the experiment of virus injection,\u0026nbsp;Morris water maze (MWM) experiments,\u0026nbsp;Barnes maze,\u0026nbsp;Fear Conditioning,\u0026nbsp;Genotyping; Xiaozhen Gu counted the cell number and\u0026nbsp;Immunohistochemistry; Chenqing Huang performed\u0026nbsp;Golgi-Cox staining and spine density assay.\u0026nbsp;All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Science Foundation of China (No. 82073592, 81773475, 81673624, 82274124，82273666, 82204084), the National Key Basic Research Program of China (No. 2018YFC1602201, 2018YFC1602204, 2012CB525003), the Key Laboratory of Xin’an Medicine Ministry of Education, Anhui University of Chinese Medicine (No. 2018xayx01).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data that support the findings of this study are available from the corresponding author, upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eLead exposure in children: prevention, detection, and management. 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Pb exposure induces an imbalance of excitatory and inhibitory synaptic transmission in cultured rat hippocampal neurons. Toxicol In Vitro 63, 104742.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"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":"Lead (Pb), neural circuit, learning and memory, virus tracing","lastPublishedDoi":"10.21203/rs.3.rs-3061407/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3061407/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eLead (Pb) is an environmental neurotoxic metal. Chronic Pb exposure causes behavioral changes in humans and rodents, such as dysfunctional learning and memory. Nevertheless, it is not clear whether Pb exposure disrupts the neural circuit. Thus, here we aim at investigating the effects the chronic Pb exposure on neural-behavioral and neural circuits in mice from prenatal to postnatal day (PND) 63. Pregnant mice and their male offspring were treated with Pb (150 ppm) until postnatal day 63. In this study, several behavior tests and Golgi-Cox staining methods were used to assess spatial memory ability and synaptogenesis. Virus-based tracing systems and immunohistochemistry assays were used to test the relevance of chronic Pb exposure with disrupted neural circuits. The behavioral experiments and Golgi-Cox staining results showed that Pb exposure impaired spatial memory and spine density in mice. The virus tracing results revealed that Entorhinal cortex (EC) neurons could be directly projected to CA1 and DG, forming a critical circuit inhibited, in either a direct or indirect way, by Pb invasion. In addition, excitatory neural input from EC(labeled with CaMK2)to CA1/DG was significantly attenuated by Pb exposure. In conclusion, our data indicated that Pb significantly impaired the excitatory connections from EC to the hippocampus (CA1 and DG), providing a novel neuro-circuitry basis for Pb neurotoxicity.\u003c/p\u003e","manuscriptTitle":"Chronic Pb exposure impairs learning and memory abilities by inhibiting excitatory projection neuro-circuit of the hippocampus in mice","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-06-26 19:10:16","doi":"10.21203/rs.3.rs-3061407/v1","editorialEvents":[{"type":"communityComments","content":0}],"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":"b5e9fdd6-9bf8-48c0-8537-7856ae8d3842","owner":[],"postedDate":"June 26th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2023-07-13T03:44:25+00:00","versionOfRecord":[],"versionCreatedAt":"2023-06-26 19:10:16","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3061407","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3061407","identity":"rs-3061407","version":["v1"]},"buildId":"WrCJVZZCHTDjtuVLN7oU0","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}