Lipid signaling determinants of physiology and pathology along the longitudinal hippocampal axis

ii DIREITOS DE AUTOR E CONDIÇÕES DE UTILIZAÇÃO DO TRABALHO POR TERCEIROS Este é um trabalho académico que pode ser utilizado por terceiros desde que respeitadas as regras e boas práticas internacionalmente aceites, no que concerne aos direitos de autor e direitos conexos. Assim, o presente trabalho pode ser utilizado nos termos previstos na licença abaixo indicada. Caso o utilizador necessite de permissão para poder fazer um uso do trabalho em condições não previstas no licenciamento indicado, deverá contactar o autor, através do RepositóriUM da Universidade do Minho. Atribuição-NãoComercial-SemDerivações CC BY-NC-ND https://creativecommons.org/licenses/by/4.0/ iii Agradecimentos/Acknowledgements À minha família, sempre. Aos meus pais e irmão pelo carinho incondicional e apoio sempre presentes. À minha mãe por ser o exemplo de força maior, ao meu pai por ser o exemplo de coragem e perseverança e ao meu irmão por nunca deixar que eu não acreditasse em mim. Ao meu orientador, Professor Doutor Tiago Gil Oliveira, que me acompanhou desde as aulas de anatomia até este doutoramento. Obrigada pelo incentivo no princípio, meio e fim desta caminhada. Obrigada pelo apoio incondicional e pelo telefonema sempre disponível, científico ou não. Ao Doutor Vítor Pinto, que muito me ensinou sobre eletrofisiologia e sobre ser pessoa. À equipa TGO que muito me deu. Obrigada especial à Francisca Vaz Bravo, Isabel Castanho, André Miranda e Rafaela Ribeiro pela ajuda, integração e companhia. Ao Torcato Meira, meu amigo que me ensinou acerca de CA2 e memória social. Aos que estiveram sempre por perto, aos “Milões”, à Marisa, ao Carlos, ao André Viana e ao João Pereira. Aos amigos que o doutoramento me trouxe e que comigo partilharam sucessos, dificuldades e cafés. Obrigada especial à Sónia Guerra Gomes, Tiago Rosa, Cláudia Antunes, Vanessa Sardinha, Jorge Silva, Diana Nascimento, João Viana, Sara Silva, Sofia Neves e Marco Guimarães. À Tuna de Medicina da Universidade do Minho, minha família durante a minha passagem na Escola de Medicina da Universidade do Minho. Aos constituintes da minha comissão de tese, Doutor Carlos Bandeira Duarte, Doutora Patrícia Monteiro e Doutor João Carlos Sousa pelas discussões científicas sempre relevantes. À Escola de Medicina da Universidade do Minho/Instituto de Investigação em Ciências da Vida e Saúde, que me acolheu enquanto aluna de Mestrado em Medicina e me proporcionou as condições para me tornar aluna de Doutoramento em Medicina. Ao João, o meu porto seguro. This work was supported by Programa Operacional Regional do Norte (NORTE2020) (fellowship UMINHO/BD/52/2017) and the BIAL Foundation, Portugal (253/14) and by national funds, through the Foundation for Science and Technology (FCT) - project UIDB/50026/2020 and UIDP/50026/2020; and by the projects NORTE-01-0145-FEDER-000013 and NORTE-01-0145-FEDER000023, supported by Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF). iv STATEMENT OF INTEGRITY I hereby declare having conducted this academic work with integrity. I confirm that I have not used plagiarism or any form of undue use of information or falsification of results along the process leading to its elaboration. I further declare that I have fully acknowledged the Code of Ethical Conduct of the University of Minho. v Determinantes lipídicos na fisiologia e patologia do eixo longitudinal do hipocampo Resumo O hipocampo é uma estrutura do lobo temporal do cérebro presente em todos os mamíferos, fundamental para a aprendizagem, memória e navegação espacial. A sua arquitetura transversal é constituída pelo giro denteado e Cornu Ammonis (CA), que se divide em CA1, CA2 e CA3, e é preservada ao longo do eixo longitudinal e em todas as espécies. Os lípidos fazem parte dos principais constituintes do cérebro e os distúrbios no seu conteúdo e metabolismo têm sido propostos como sendo de particular importância em várias doenças. A análise por espectrometria de massa das porções dorsal (HD), intermédia e ventral (HV) do hipocampo revelou um gradiente lipídico contínuo ao longo do eixo longitudinal, com maior distinção entre o HD e HV. O HD apresenta níveis superiores de ácido fosfatídico e níveis reduzidos de fosfatidilcolina em comparação com o HV, potencialmente implicando a via da fosfolipase D (PLD) na regulação do eixo HD-HV. A superfamília da PLD possui seis membros (PLD1-PLD6), mas apenas a PLD1 e a PLD2 hidrolisam fosfatidilcolina em ácido fosfatídico. O nosso principal objetivo foi estudar o impacto da modulação genética da PLD na organização e função do eixo HD-HV. Em primeiro lugar, mostramos que a PLD1 e a PLD2 são os únicos contribuintes para a atividade total da PLD no cérebro de ratinho, embora a PLD1 seja uma fonte maioritária de ácido fosfatídico no HD e HV em comparação com a PLD2. A seguir, abordamos como a PLD1 afeta diferencialmente a organização e o funcionamento do hipocampo em cada subregião. A ablação da PLD1 afeta predominantemente o lipidoma do HD e os ratinhos Pld1 knockout (KO) apresentam déficits específicos no reconhecimento de objetos novos e interação social e também disrupção na diferenciação da arborização dendrítica do HD e HV em neurónios piramidais de CA1/CA3. Apresentam ainda diminuição na indução de depressão de longo-prazo (LTD) e redução dos níveis das proteínas GluN2A e SNAP-25 no HD. De seguida, abordamos os efeitos da ablação PLD2 no comportamento social e na potenciação sináptica hipocampal. Mostramos que a ablação PLD2 leva a défices de discriminação social, nomeadamente no reconhecimento de ratinhos da mesma ninhada, diminuição da exploração social e redução de potenciação de longo-prazo (LTP) no HV. Com nosso trabalho mostramos como a PLD regula o hipocampo e a sua função, mostrando que, embora a PLD1 e a PLD2 catalisem a mesma reação, a sua ablação específica promove fenótipos diferentes. Isto tem possíveis implicações em doenças neurodegenerativas, como a doença de Alzheimer, na qual PLD tem um papel importante para a sua patologia. Palavras-chave: Fosfolipase D, Hipocampo, Lípidos, Memória vi Lipid signaling determinants of physiology and pathology along the longitudinal hippocampal axis Abstract The hippocampus is a brain temporal lobe structure present in all mammals fundamental for learning, memory and spatial navigation. Its cross-sectional architecture, constituted by the dentate gyrus (DG) and Cornu Ammonis (CA) subfields, CA1, CA2 and CA3, is preserved along the longitudinal axis and in every species. Lipids are major constituents of the brain and disturbances in brain lipid content and metabolism have been proposed to be of particular importance in several diseases. A mass spectrometry analysis of dorsal (DH), intermediate and ventral (VH) portions of the hippocampus revealed a continuous lipid gradient along the longitudinal axis, with greater distinction between DH and VH. DH presents increased phosphatidic acid (PA) and decreased phosphatidylcholine (PC) compared to the VH, potentially implicating the phospholipase D (PLD) pathway in DH-VH axis regulation. The PLD superfamily has six members (PLD1-PLD6), but only PLD1 and PLD2 hydrolyze PC into PA. Our main goal was to study the impact of PLD genetic modulation on the DH-VH axis organization and function. First, we showed that PLD1 and PLD2 are the only contributors to total PLD activity in the mouse brain, although PLD1 is a major source of PA in both DH and VH when compared to PLD2. Next, we addressed how PLD1 differentially affects hippocampal organization and functioning in a region-specific manner. PLD1 ablation affects predominantly the lipidome of the DH and Pld1 knockout (KO) mice present specific deficits in novel object recognition and social interaction, disruption in the DH-VH dendritic arborization differentiation in CA1/CA3 pyramidal neurons, along with reduced longterm depression (LTD) induction and reduced GluN2A and SNAP-25 protein levels in the DH. Then, we address the effects of PLD2 ablation in social behavior and hippocampal synaptic potentiation. We showed that PLD2 ablation leads to social discrimination deficits, namely littermate recognition impairment, decreases social exploration and reduces long-term potentiation (LTP) in the VH. With our work we show how PLD differentially regulates the hippocampus, showing that, although PLD1 and PLD2 catalyze the same reaction, their specific ablation promotes different phenotypes. This leads to potential implications in neurodegenerative diseases, such as Alzheimer’s disease, in which PLD is an important player. Keywords: Hippocampus, Lipids, Memory, Phospholipase D vii Table of Contents Resumo ............................................................................................................................................................ v Abstract ............................................................................................................................................................ vi Abbreviations List .............................................................................................................................................. ix Figures, Tables and Boxes ............................................................................................................................... xiii CHAPTER 1: General Introduction ...................................................................................................................... 1 1.1. Hippocampus ............................................................................................................................. 2 1.2. Synaptic Plasticity ....................................................................................................................... 7 1.3. Lipids ........................................................................................................................................ 9 1.4. Phospholipids and Phospholipases ............................................................................................. 11 1.5. Phospholipase D ....................................................................................................................... 13 1.6. Phosphatidic Acid Signaling ....................................................................................................... 20 1.7. Aims of the thesis ..................................................................................................................... 22 References ........................................................................................................................................... 23 CHAPTER 2: Phospholipase D1 Ablation Disrupts Mouse Longitudinal Hippocampal Axis Organization and Functioning ..................................................................................................................................................... 35 Abstract ............................................................................................................................................... 37 Introduction.......................................................................................................................................... 37 Results ............................................................................................................................................... 38 Discussion ........................................................................................................................................... 41 References ........................................................................................................................................... 46 Materials and Methods .......................................................................................................................... 49 CHAPTER 2 – Supplementary Information ....................................................................................................... 55 CHAPTER 3: Phospholipase D2 ablation leads to deficits in social memory ....................................................... 64 Abstract ............................................................................................................................................... 65 Introduction.......................................................................................................................................... 66 Materials and Methods .......................................................................................................................... 68 Results ............................................................................................................................................... 73 xiv Supplementary Figure 3. No compensation at protein levels upon PLD1 or PLD2 ablation. Related to Figure 1. Supplementary Figure 4. Supplementar behavior analysis. Related to Figure 2. Supplementary Figure 5. PLD1 ablation has no effect in input-output recordings. Related to Figure 4. Table 1. Descriptive statistics of LC-MS analysis of lipid classes of DH and VH from Pld1 KO and Pld2 KO animals, related to Figure 1. Table 2. Descriptive statistics of LC-MS analysis of PA species of DH and VH from Pld1 KO and Pld2 KO animals, related to Figure 1. Chapter 3 Figure 1. PLD2 ablation impairs the ability to recognize a littermate and decreases social exploration. Figure 2. PLD2 ablation impairs the ability to generate LTP in VH. Figure 3. PLD2 ablation has no greater impact on synaptic proteins. Chapter 3 – Supplementary Information Figure S1 – Supplementary behavior analysis. Related to Figure 1 Figure S2 – PLD2 ablation has no effect in input-output recordings. Related to Figure 2. Chapter 4 Box 1. Chapter 2 highlights. Box 2. Chapter 3 highlights. 1 CHAPTER 1 General Introduction 2 1.1. Hippocampus The hippocampus is a brain temporal lobe structure present in all mammals fundamental for learning, memory and spatial navigation (Strange et al., 2014). Classically, memory can be divided in short-term (working) memory and long-term memory, which, in its turn, can be subdivided in non-declarative (implicit) memory and declarative (explicit) memory (see Figure 1). Both episodic (memory for events or episodes in a specific spatial and temporal context) and semantic memory (generic memory about people, objects, places, new word meanings and the world in general) compose the declarative long-term type of memory (Kandel et al., 2000). The processing of declarative memory involve encoding (where new information is attended and linked to existing information in memory), storage (mechanism by which memory is retained over time), consolidation (the mechanism that makes the temporarily stored and still labile information more stable, which implicates expression of genes and protein synthesis that give rise to structural changes at synapses) and retrieval (the process by which stored information is recalled) (Kandel et al., 2000). Figure 1. Types of memory. The hippocampal role in declarative memory was first considered with the work of Scoville and Milner with the patient H.M., after a bilateral temporal resection in an attempt to treat temporal lobe epilepsy (Scoville and Milner, 1957). As a result, the seizures were controlled but severe anterograde amnesia emerged as a side effect, with deficient acquisition of episodic and semantic knowledge (Corkin, 2002). 3 The hippocampal role in spatial navigation was contemplated with the discovery of “place cells”, which are pyramidal neurons that become activate when a subject is in a particular environment or “place field” (O'Keefe and Dostrovsky, 1971; O'Keefe and Speakman, 1987), and with the realization that hippocampal lesions cause a profound and lasting impairment in place-navigation (Morris et al., 1982). Later, entorhinal grid cells were also proposed to encode the spatial map (Hafting et al., 2005). These neurons fire in hexagonal grid patterns and transmit information to place cells (Hafting et al., 2005). Conjointly, these two types of cells from the hippocampal and parahippocampal formations work to grant positioning awareness and spatial representations (Moser et al., 2008). The hippocampus is curved-shaped and its cross-sectional architecture is preserved along the longitudinal axis and in every species (Strange et al., 2014). The hippocampal formation consists of dentate gyrus (DG), hippocampus proper – Cornu Ammonis (CA), consisting of CA1, CA2 and CA3 – and subiculum (Strange et al., 2014; van Strien et al., 2009). It has a three-layered appearance histologically. The deep layer, constituted by afferent and efferent fibers and interneurons, is the hilus in the DG and the stratum oriens in the CA (van Strien et al., 2009). The intermediate layer is the cell layer, mainly with cells and interneurons, and it is called granule layer (with granular cells that present only apical dendrites in mice) in the DG and stratum pyramidale (with pyramidal neurons that express both apical and basal dendrites) in the CA and the subiculum (Kandel et al., 2000; van Strien et al., 2009). The superficial layer is called the stratum moleculare in the DG and in the subiculum (van Strien et al., 2009). In CA3, the superficial layer is subdivided in three: the stratum lucidum, the stratum radiatum and the stratum lacunosum-moleculare (van Strien et al., 2009). The first receives input from the DG; the second shelters the apical dendrites from stratum pyramidale neurons; and the last comprises the apical tufts of the apical dendrites (van Strien et al., 2009). In CA1 and CA2 the stratum lucidum is missing (van Strien et al., 2009). All these structures interconnect in complex intrahippocampal circuits. The entorhinal cortex (EC) is a hippocampal nearby structure that sends sensory and spatial information to the hippocampus through the perforant direct and indirect pathways (see Figure 2) (Kandel et al., 2000; Strange et al., 2014; van Strien et al., 2009). The monosynaptic direct pathway is originated in the layer III of the EC and its neurons form synapses on the distal apical dendrites of CA1 neurons (Kandel et al., 2000; van Strien et al., 2009). The indirect pathway has its origins in the layer II of the EC and reaches the more proximal regions of CA1 through the 4 trisynaptic pathway: neurons from layer II of EC synapse onto DG granule cells (synapse 1) whose axons project in the mossy fiber pathway to excite the pyramidal cells in the CA3 (synapse 2) and then axons of the neurons from CA3 region project through the Schaffer collateral pathway to make excitatory synapses on CA1 pyramidal cell dendrites (synapse 3) (Kandel et al., 2000; van Strien et al., 2009). Both pathways are necessary for normal learning and memory. Inactivation of the indirect pathway impairs the competence to perform complex spatial learning and memory tasks while lesions of the direct pathway impair episodic memory and interfere with the ability to consolidate memory (Kandel et al., 2000). The major output of the hippocampus are the pyramidal neurons in CA1 region, which have projections to the layer V of EC and to the subiculum (which also projects to layer V of the EC), closing the circuit (Kandel et al., 2000). Regarding EC connections, it is known that cells from EC layer II also project to CA3 (Tamamaki and Nojyo, 1993) and that neurons from EC layer III project to the subiculum in addition to CA1 (direct pathway) (Honda et al., 2012). Figure 2. Classical cortico-hippocampal synaptic circuitry. Schematic representation of the intrahippocampal connectivity and connection with EC. The trisynaptic pathway is highlighted in red and the direct (monosynaptic) pathway is highlighted in blue. CA, Cornu Ammonis; DG, dentate gyrus; EC, entorhinal cortex. Afterward, studies focusing on CA2 revealed other cortico-hippocampal pathways. CA2 pyramidal neurons receive strong input from layer II of the EC and are weekly activated by CA3 neurons. In 5 their turn, CA2 neurons strongly activate CA1 neurons (Chevaleyre and Siegelbaum, 2010). Thus, CA2 provides a disynaptic link between the cortical input to the hippocampus to its CA1 neuronal output (Chevaleyre and Siegelbaum, 2010). Moreover, DG neurons form monosynaptic inputs to CA2 pyramidal neurons (Kohara et al., 2014). In addition, there are CA3-CA2 connections dominated by feedforward inhibition (Chevaleyre and Siegelbaum, 2010; Kohara et al., 2014). Inhibition or genetic lesions of each of these structures improved the knowledge about their function. Deletion of N-methyl-D-aspartate receptor 1 (GluN1, also known as the NMDAR1 or NR1) in CA1 pyramidal neurons leads to impaired spatial memory, because of impairment in CA1 long-term potentiation (LTP) (see below), without disruption of nonspatial learning (Tsien et al., 1996). Dorsal CA1 and subiculum are involved in contextual memory (Goshen et al., 2011; Roy et al., 2017) and inhibition of dorsal CA1, sparing other hippocampal regions, such as dorsal DG and CA3 and ventral CA1, impairs remote memory recall (Goshen et al., 2011). Dorsal CA1 inhibition with muscimol, a selective GABAA receptor agonist, also impairs spatial and object memory (Stackman et al., 2016). On the other hand, ventral CA1 (and its projections to the nucleus accumbens) is implicated in the storage of social memory (Okuyama et al., 2016). In fact, dorsal CA2 projections to ventral CA1 are necessary for social memory (Meira et al., 2018). Deletion of GluN1 in CA3 pyramidal cells also leads to deficits in social memory and, ventral, but not dorsal, CA3 is necessary for the encoding of a social memory (Chiang et al., 2018). Targeted manipulation of GluN receptors in CA3 showed that the output from CA3 in the trisynaptic pathway is dispensable for spatial learning, but the full trisynaptic pathway containing CA3 was required for rapid one-trial contextual learning and for pattern completion recall (Nakashiba et al., 2008). The DG is a brain region where neurogenesis persists into adulthood (Altman and Das, 1965; Anacker and Hen, 2017; Hochgerner et al., 2018). A transgenic mouse model in which output of old granule cells was specifically inhibited while leaving a substantial portion of young granule cells intact displayed enhanced or normal contextual and spatial pattern separation (ability to distinguish between two similar contexts), which was reduced following ablation of young granule cells. In addition, these animals exhibited deficits in contextual and spatial pattern completion (ability to recall a memory based on incomplete information). This indicates that pattern separation depends on adult-born young granule cells, while recall by pattern completion needs older granule cells (Nakashiba et al., 2012). 6 Genetic ablation of layer III inputs from EC to the hippocampus leads to an impairment in hippocampal-dependent temporal association memory (the ability to associate temporally discontinuous elements) assessed by trace fear conditioning (Suh et al., 2011). Despite the preservation of the same basic intrinsic circuit, anatomical features (such as cortical and subcortical connections) and electrophysiological, gene expression and functional studies distinguish a longitudinal dorsal-ventral (DH-VH) axis in rodents, that segregate into DH-VH poles, which correspond in primates to posterior-anterior poles, respectively (Strange et al., 2014). Despite the sharply demarcated dichotomous segregation into dorsal-ventral portions, it has also been enrolled a gradual biochemical and functional transition between the two poles (Strange et al., 2014). The classical dichotomic view associates predominantly the DH in spatial navigation and episodic memory and the VH in emotional stress-related behaviors (Bannerman et al., 2014; Fanselow and Dong, 2010; Kheirbek et al., 2013; McHugh et al., 2011; Strange et al., 2014) and also in motivational behaviors because of its connections with the amygdala and hypothalamus (see Figure 3) (van Strien et al., 2009). On the other hand, DH-VH organization of extrinsic connectivity follows topographically organized projections along the hippocampus (Strange et al., 2014). Figure 3. Hippocampus dichotomic view. Expression of specific genes also support this long-axis organization (Strange et al., 2014), for example, NMDAR expression decreases from dorsal to ventral in CA1 (Maruki et al., 2001) and hippocampal gene expression analysis predicted functional differentiation across the full longitudinal axis of the hippocampus (Bienkowski et al., 2018; Shah et al., 2017; Thompson et 7 al., 2008). Also, low-affinity glucocorticoid receptors are more expressed in the DH, while highaffinity mineralocorticoid receptors are more expressed in the VH (Segal et al., 2010). Chronic unpredictable stress also has a different impact along the DH-VH axis of the hippocampus. In the DH, stress triggers a volumetric reduction as a result of atrophy of CA3 and CA1 apical dendrites, whereas in the VH there is an increase in hippocampal volume concurrent with the increase of CA3 apical dendrites. Moreover, electrophysiological data revealed that stress leads to a decrease in VH long-term depression (LTD) (see below) (Maggio and Segal, 2007; Pinto et al., 2015). Regarding other electrophysiological features, when dividing the hippocampus along its septotemporal axis into five sections, LTP is specifically reduced in the more ventral portion, compared with the rest of the hippocampus (Maggio and Segal, 2007). This was reversed by an exposure to acute stress, allowing VH to express a large LTP, whereas in the rest of the hippocampus it was much suppressed (Maggio and Segal, 2007). Nonetheless, LTD in VH is of similar magnitudes as the ones expressed in DH (Pinto et al., 2015). Short-term plasticity is also different between DH and VH, with DH presenting higher levels of paired pulse (PP) facilitation (Maruki et al., 2001; Pinto et al., 2015). Concerning lipid composition, a broad-scale analysis of dorsal, intermediate and ventral portions of the hippocampus revealed a continuous gradient along the longitudinal axis, with greater distinction between DH and VH (Miranda et al., 2019). Treatment with corticosterone in rats also led to region-specific modulation of lipid species, displaying VH as more sensitive to corticosterone (Miranda et al., 2019). Altogether, these findings provide insight for hippocampal organization and functioning in a region-specific manner. 1.2. Synaptic Plasticity One of the most important characteristics of the mammalian brain is its plasticity. It has been proposed that associative memories are stored as changes in the strength of synaptic connections between neurons, also called synaptic plasticity (Bannerman et al., 2014; Citri and Malenka, 2008). Numerous forms of synaptic plasticity have been characterized. Synaptic 8 transmission can last from milliseconds to several minutes (short-term synaptic plasticity) or longer (long-term synaptic plasticity) (Citri and Malenka, 2008). Short-term plasticity is important for short-term adaptations to sensory inputs, transient changes in behavioral states and short-lasting forms of memory (Citri and Malenka, 2008). It is triggered by short bursts of activity, causing a transient accumulation of calcium in presynaptic nerve terminals, leading to changes in the probability of neurotransmitter release (Citri and Malenka, 2008). When two stimuli are delivered within a short interval, the response to the second stimulus can be enhanced or depressed relative to the response to the first stimulus – PP facilitation or depression. One synapse can display either facilitation or depression depending on the previous activation or modulation (Citri and Malenka, 2008). PP depression occurs within short interstimulus intervals (less than 20ms) and can be caused by depletion of some pool of readily releasable vesicles (Zucker and Regehr, 2002) or it can arise from feedback activation of presynaptic receptors and from postsynaptic processes such as receptor desensitization (Citri and Malenka, 2008; Zucker and Regehr, 2002). Longer interstimulus intervals (20–500ms) are responsible for PP facilitation, since the residual calcium left over from the invasion of the first action potential contributes to additional release during the second stimulation (Zucker and Regehr, 2002) or due to the activation of protein kinases that regulate the activity of presynaptic phosphoproteins (Citri and Malenka, 2008). Longer-lasting forms of plasticity can be seen after tetanic stimulation with prolonged (200ms to 5s) trains of stimulation applied at high frequencies (10-200 Hz) – augmentation and post-tetanic potentiation (Zucker and Regehr, 2002). Memory relies on changes in synaptic strength, which can take the form of persistent enhancements, such as LTP of LTD (Bear, 1996; Kemp and Manahan-Vaughan, 2004). Most hippocampal studies regarding long-term plasticity are focused on LTP in CA1, but it is known that the majority of synapses that exhibit LTP also exhibit LTD (Citri and Malenka, 2008). A brief high-frequency train of stimuli gives rise to LTP and distinct forms of LTP diverge in the relative importance of diverse receptors and ion channels and each one may be able to recruit distinct second-messenger signaling pathways (Kandel et al., 2000). Postsynaptic NMDAR are necessary for LTP at the Schaffer collateral, mossy fiber and entorhinal inputs to CA1, although its contribution diverges in the three pathways (Kandel et al., 2000). Therefore, inhibition of LTP in the hippocampus interferes with spatial memory (Kandel et al., 2000). LTP can also be split into a short and long-term phases, this last requiring the synthesis of new proteins and mRNA for its persistence (Kauderer and Kandel, 2000). 9 LTD is a mechanism that downregulates synaptic function and it was first identified in the cerebellum, with relevance for motor learning (Kandel et al., 2000). LTD induction requires longer periods of low-frequency synaptic stimulation (Kandel et al., 2000) and can also be divided in phases, depending an early-phase of covalent modifications of preexisting proteins, whereas a late-phase requires transcription and translation (Kauderer and Kandel, 2000). 1.3. Lipids Lipids can be defined as fat-soluble molecules based on their solubility in non-polar solvents (Lam and Shui, 2013). Lipids are a naturally occurring group of amphipathic molecules, which means they hold both a hydrophobic/apolar domain and a hydrophilic domain, which interacts with water (Simons and Sampaio, 2011). This hydrophobic effect results from the hydrocarbon domains that distort the stable hydrogen-bonded structure of water by inducing cage-like structures around the apolar domains (Simons and Sampaio, 2011). Moreover, the lipids degree of length and saturation provides different physical properties to membranes, such as thickness, fluidity, and microdomain assembly (Holthuis and Menon, 2014). Based on their functional backbone structure, a commonly used classification divides lipids into eight categories: fatty acyls, glycerolipids, glycerophospholipids, sphingolipids, sterol lipids, prenol lipids, saccharolipids and polyketides (Fahy et al., 2009). Fatty acyls possess a carboxylic group attached to an aliphatic chain; saturated fatty acids do not contain double bonds, whereas monounsaturated and polyunsaturated fatty acids contain one or more cis double bonds. Glycerolipids are fatty acid esters of glycerol and comprise mono-, diand tri-acylglycerols. Glycerophospholipids contain phosphoric acid in ester form with a glycerolipid and are subdivided into distinct classes based on the nature of the head group linked to the phosphate. Sphingolipids contain a common sphingoid base backbone and are acylated to form ceramides, which are modified to generate phosphosphingolipids and glycosphingolipids. Sterol lipids, of which cholesterol and its derivatives are the most widely studied in mammalian systems, contain a fused four-ring core and are subdivided based on the number of carbons in the core skeleton. Prenols are synthesized from the five carbon precursors isopentenyl diphosphate and dimethylallyl diphosphate that are produced mainly via the mevalonic acid pathway. Saccharolipids are compounds in which fatty acids are linked directly to a sugar backbone (the sugar substitutes for the glycerol backbone that is present in glycerolipids and 16 activation of small GTPases, such as Ras homolog family member A (RhoA) and ADP ribosylation factor (ARF) family members, and protein kinase C (PKC). Other regulatory mechanisms including phosphorylation, availability of the lipid cofactor PI(4,5)P2, and other protein activators have been proposed (Bruntz et al., 2014; Jenkins and Frohman, 2005; Salazar and Frohman, 2020). With the emergence genetic PLD mouse models, namely Pld1 knock-out (KO) (Elvers et al., 2010) and Pld2 KO (Oliveira et al., 2010) mice, it was possible to study the mammalian phenotypes upon PLD ablation. Studies in Pld1 and/or Pld2 KO mice result in homozygous viable adults, although it has been reported by some authors that Pld1 KO and Pld2 KO animals present increased body weight and body fat content, accompanied by an increased food intake (Trujillo Viera et al., 2016). Other reported observations mentioned PLD1 and/or PLD2 deficient mice to have reduced brain growth at 14-27 days after birth, but that is eventually caught up with WT mice at day 33 (Burkhardt et al., 2014). Moreover, Pld2 KO mice have an increased number of ectopic cerebellar Purkinje cells and olfactory defects after 13 weeks of age (Vermeren et al., 2016). Regarding mice behavior, conflicting results have been reported upon PLD1 or PLD2 ablation. Oliveira et al. reported that Pld2 KO mice have no memory or learning deficits when compared to control mice (Oliveira et al., 2010). However, Burkhardt et al. showed a deficit at the level of discrimination in long-term novel object recognition test in Pld2 and double Pld KO and a social recognition deficit in Pld1 and/or Pld2 KO (Burkhardt et al., 2014). The hallmarks of AD are the extracellular amyloid beta (Aβ) deposition (generated from amyloid precursor protein (APP) by sequential cleavages mediated by the β-secretase β-site APP cleavage enzyme 1, BACE1, and the γ-secretase complex) as senile plaques and the intracellular accumulation of hyperphosphorylated tau as neurofibrillary tangles (Di Paolo and Kim, 2011). Several studies have addressed the link between PLD1/PLD2 and AD, either in Aβ signaling (Bravo et al., 2018; Oliveira et al., 2010) and amyloid precursor protein APP processing and trafficking (Cai et al., 2006a; Cai et al., 2006b). Kanfer et al. found an increase in PLD activity in AD brain extracts (Kanfer et al., 1996). PLD1 has been implicated in APP processing and trafficking. For instance PLD1 has been shown to regulate the intracellular trafficking of presenilin 1(PS1)/γ-secretase (Liu et al., 2009). Moreover, it has been suggested that reducing the APP trafficking back to the trans-Golgi network results in an increase of amyloidogenesis and several studies implicate PLD1 as well as its product PA in the budding of secretory vesicles from the 17 trans-Golgi network (Chen et al., 1997; Ktistakis et al., 1995; Riebeling et al., 2009). In fact, Cai et al. showed that PS1 physically interacts with PLD1, recruiting it to the Golgi complex/transGolgi network (Cai et al., 2006a). Also, overexpression of PLD1 decreases the levels of Aβ whereas silencing PLD1 produces the opposite effect (Cai et al., 2006a). In a companion publication, PLD1 overexpression was shown to promote the formation of APP-containing secretory vesicles from trans-Golgi network and inhibition of PLD1 activity decreases APP trafficking (Cai et al., 2006b). PLD1 is able to correct the impaired APP trafficking and neurite outgrowth in familiar AD-linked PS1 mutant neurons (Cai et al., 2006b). Aberrant elevated synaptosomal PLD1 was observed in AD hippocampi compared to age-matched controls and this elevated PLD1 plays a key detrimental role in facilitating both Aβ and tau oligomer-driven synaptic dysfunction and underlying memory deficits (Krishnan et al., 2018) and, also, the use of a PLD1 inhibitor (VU0 155069) is sufficient to prevent the progression of synaptic dysfunction during early stages in the 3xTg-AD mouse model (Bourne et al., 2019). Oliveira and colleagues showed that Aβ oligomers induce total PLD activity in primary cortical neurons, which is abolished in mouse cultured neurons lacking PLD2, and that genetic ablation of PLD2 leads to attenuation of AD pathogenesis by increasing resistance to Aβ oligomer insult and memory deficit protection in the SwAPP mouse model (Oliveira et al., 2010). In addition to these findings relating PLD to AD, it is also known that synucleins interact with and inhibit PLD (Ahn et al., 2002; Jenco et al., 1998; Payton et al., 2004). Moreover, ablation of these enzymes promotes other phenotypes regarding autophagy (Dall'Armi et al., 2010), tumor biology (Chen et al., 2012; Wang et al., 2017), neurobiology (Ammar et al., 2015; Oliveira et al., 2010; Vermeren et al., 2016) and platelet function (Elvers et al., 2010; Thielmann et al., 2012). Canonical enzymatic activity has not been described for non-classical PLDs, such as PLD3, PLD4, PLD5 and PLD6. These members lack the two functional domains PX and PH which are found in the N-terminal regions of PLD1 and PLD2 and instead contain a putative transmembrane (TM) domain (Yoshikawa et al., 2010). PLD3 ( Hu-K4 in humans, SAM-9 in murines) and PLD4 are 5’ exonucleases glycosylated type II transmembrane proteins (Fazzari et al., 2017; Gavin et al., 2018; Gonzalez et al., 2018; Otani et al., 2011). PLD3 is a located in endoplasmatic reticulum and endolysosomes (Frohman, 2015; Gavin et al., 2018; Munck et al., 2005; Osisami et al., 2012). In 2014, a genome wide-association study identified a rare variant of PLD3 (Val232Met) that doubled the risk for late-onset AD. In addition, two other PLD3 variants, 18 Ala442Ala and Met6Arg, were reported to be nominally associated with AD risk. This study showed that PLD3 is highly expressed in the hippocampus and cortex, brain regions vulnerable to AD pathology, and that AD brains have lower levels of PLD3 in their neurons (Cruchaga et al., 2014). Moreover, Cruchaga et al. observed that overexpression of PLD3 leads to a significant decrease in intracellular APP and extracellular Aβ42 and Aβ40 and PLD3 knockdown had an opposite effect (Cruchaga et al., 2014). These data led to propose PLD3 loss-of-function as an AD risk factor by affecting APP processing but, although there are studies in agreement with some of these data (van der Lee et al., 2015), several reports have questioned some of the findings regarding molecular association between PLD3 and AD (Hooli et al., 2015; Lambert et al., 2015). In addition, Fazzari et al. observed no alterations in full-length APP, C-terminal APP fragments and the ratio between full-length and C-terminal fragments expression under genetic deletion of Pld3 . In addition, they found that PLD3 was localized in late and endosomes and lysosomes (visualized with the lysosomal-associated membrane protein 1 (LAMP1) marker) and lysosomes from PLD3-deficient CA1 neurons displayed an increase in density, size and in total area occupied with lipid droplets inclusions (Fazzari et al., 2017). Later, another study showed that PLD3 is a lysosomal protein that becomes proteolytically cleaved in acidic compartments and inhibitors of intracellular trafficking or lysosomal acidification abolished this processing (Gonzalez et al., 2018). Gonzalez et al. also proposed that the biosynthetic route of PLD3 depends on the endosomal sorting complex required for transport (ESCRT) machinery to reach lysosomes in mammalian cells (Gonzalez et al., 2018). In 2019 two other rare variants of Pld3 were described in late-onset AD, which could lead to reduced PLD3 activity and affect Aβ levels in cellular model of AD, via autophagy-dependent mammalian target of rapamycin (mTOR) pathway (Tan et al., 2019). Since disruption of endolysomal membrane trafficking flux has been shown to lead to neurodegeneration and impairment in APP trafficking and processing (Miranda et al., 2018), the role of PLD3 in lysosome functioning is a strong biological hypothesis for the reported human genetic association in AD (Cruchaga et al., 2014). PLD4 is localized in organelle membranes, including the endoplasmatic reticulum and Golgi complex (Yoshikawa et al., 2010) and also in phagosomes (Otani et al., 2011). Its biological function has not been fully elucidated, but some studies associate PLD4 to autoimmune diseases. Pld4 risk allele was associated with anti-double stranded DNA antibodies production, implying PLD4 contribution to systemic lupus erythematosus by hyperactivating B-cells (Akizuki et al., 2019). Pld4 mutant mice have low body weight and also autoimmune phenotypes 19 corresponding to systemic lupus erythematosus, including nephritis (Akizuki et al., 2019). Moreover, it has been demonstrated that PLD4 promotes kidney fibrogenesis by modulating innate and adaptative immune responses (Trivedi et al., 2017). Other studies linked PLD4 polymorphisms to systemic sclerosis (Terao et al., 2013) and rheumatoid arthritis (Okada et al., 2012). Lastly, Gavin et al. presented evidence that the endolysosomal proteins PLD3 and PLD4 are 5′ exonucleases that break down toll-like receptor (TLR) 9 ligands, thus degrading them and limiting their ability to stimulate TLR9. Hence, macrophages from PLD3-deficient mice have exaggerated TLR9 responses (Gavin et al., 2018) and PLD4-deficient mice display a TLR9-driven inflammatory syndrome and splenomegaly (Chen et al., 2017; Gavin et al., 2018). PLD5 is encoded by an autosomal gene (Yao et al., 2020) and has nonconservative substitutions in its putative catalytic site, making it unlikely to be enzymatically active and definitive cellular or physiological roles have not yet been recognized (Nelson and Frohman, 2015). One study correlates a single-nucleotide polymorphism in the Pld5 gene with verbal performance in autism patients, but the clinical importance of this finding is not clear (Anney et al., 2010). PLD6 (also known as mitoPLD) encodes only one HKD motif and dimerizes to exhibit catalytic activity (Nelson and Frohman, 2015). It is anchored by an N-terminal transmembrane tail into the outer surface of mitochondria (Choi et al., 2006) and hydrolyzes cardiolipin on the outer surface of the mitochondria to generate PA (Choi et al., 2006). It also acts as an endonuclease of RNA to generate specialized microRNAs known as P-element-induced wimpy testis (piwi)-interacting RNAs (Voigt et al., 2012), that control gene modification and stability (Moyano and Stefani, 2015), which are critical during spermatogenesis (Huang et al., 2011; Kabayama et al., 2017). PLD is still a rapidly rising field of research with recent major technical advances. For instance, it was recently developed a method named IMPACT (Imaging Phospholipase D Activity with Clickable Alcohols via Transphosphatidylation), which is a chemical method for imaging phosphatidyl-alcohols synthesized by PLD enzymes in live cells, relying in azidoalcohols as reporters in a transphosphatidylation reaction (Bumpus and Baskin, 2016; Bumpus et al., 2018) and also an optogenetic PLD which allows the visualization of cellular PLD activity (Tei and Baskin, 2020). Moreover, crystal structures of PLD are emerging, either in plants (Li et al., 2020) and humans (Metrick et al., 2020), providing explanations for structural insights and promoting structure-based drug discoveries. 20 1.6. Phosphatidic Acid Signaling PA has important physiological and signaling roles, since it contributes to membrane biogenesis and is also a signaling molecule. It is composed of a three-carbon glycerol backbone, two fatty acid chains and a small phosphate headgroup, thus referred to as a “cone shape” lipid (i.e., a lipid with a small head groups relative to a large hydrophobic domain) (Cazzolli et al., 2006; Jenkins and Frohman, 2005). PA is negatively charged, promoting a negative membrane curvature and making it possible to alter the properties of the membranes (Jenkins and Frohman, 2005). PA is involved in important cellular processes such as membrane biogenesis and trafficking events and different PA species exhibit different structural properties, which is related to the acyl chain length and unsaturation of the fatty acyl chain at sn-1 and sn-2 positions, leading to distinct repercussion on membrane structure, for example membrane curvature (van Meer, 2005). In fact, PA saturation level influences its functions in neurosecretory pathway (Tanguy et al., 2020). While mono-unsaturated PA regulates the number of exocytotic events, poly-unsaturated PA regulates fusion pore stability and expansion (Tanguy et al., 2020). PA is a critical element in signaling pathways as a second messenger and it can also be further metabolized to other signaling lipids such as DAG by PA phosphatases, lysophosphatidic acid (LPA) by phospholipase A and other lipids such as PI (Oliveira and Di Paolo, 2010; Sonoda et al., 2002). On the other hand, PLD is not the only source of PA, since it can also be obtained from DAG by a reaction catalyzed by DAG kinase, from LPA as a result of LPA acyltransferases (LPAAT) activity, from cardiolipin catalyzed by mitoPLD and other enzymes in the biosynthetic pathway (see Figure 6) (Choi et al., 2006; Haucke and Di Paolo, 2007). 21 Figure 6. PA metabolism. PA can be generated from other sources and further metabolized. PA, phosphatidic acid; PLD, Phospholipase D; PC, phosphatidylcholine; LPA, lysoPA; DAG, diacylglycerol; CDP-DAG, cytidine diphosphate-DAG; PLA, phospholipase A; LPAAT, LPA acyltransferase; PAP, PA phosphatase; DGK, DAG kinase; CDS, CDP-DAG synthase; mitoPLD, mito-phospholipase D. PA plays an important role in membrane trafficking, for example, in phagocytosis (Corrotte et al., 2006; Tanguy et al., 2019), autophagy (Holland et al., 2016), synaptic transmission (Humeau et al., 2001; Raben and Barber, 2017), among others, since it may recruit specific proteins and/or facilitate fusion because of higher curvature of PA-enriched membranes (Tanguy et al., 2020). PA is crucial for many activation pathways of intracellular signaling transduction events, such as PI phosphate kinases, activation of mTOR, and direct binding with Rapidly Accelerated Fibrosarcoma (Raf) kinase, soluble N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) proteins and sphingosine kinases (Cazzolli et al., 2006; Jenkins and Frohman, 2005; Stace and Ktistakis, 2006). In fact, specific PLD-derived PA modulates subcellular processes such as vesicular transport (Cai et al., 2006b; Choi et al., 2002; Huang et al., 2005; Raghu et al., 2009a; Vitale et al., 2001), NADPH oxidase activity (Zhang et al., 2009), mTOR and S6K activity (Fang et al., 2003). 22 1.7. Aims of the thesis Recently, a mass spectrometry study revealed that the DH presents increased PA and decreased PC compared to the VH, potentially implicating the PLD pathway in DH-VH axis regulation (Miranda et al., 2019). Since the cell biology and pathophysiology involving PLD1 and PLD2 remain a topic of investigation, our main goal was to study the impact of PLD genetic modulation on the DH-VH axis organization and function. In chapter 2, we address how PLD1 ablation differentially affects hippocampal organization and functioning in a region-specific manner. In chapter 3, we address the effects of PLD2 ablation in social behavior and hippocampal synaptic potentiation. 23 References Ahn, B.H., Rhim, H., Kim, S.Y., Sung, Y.M., Lee, M.Y., Choi, J.Y., Wolozin, B., Chang, J.S., Lee, Y.H., Kwon, T.K. , et al. (2002). alpha-Synuclein interacts with phospholipase D isozymes and inhibits pervanadate-induced phospholipase D activation in human embryonic kidney-293 cells. The Journal of biological chemistry 277 , 12334-12342. Akizuki, S., Ishigaki, K., Kochi, Y., Law, S.M., Matsuo, K., Ohmura, K., Suzuki, A., Nakayama, M., Iizuka, Y., Koseki, H. , et al. (2019). PLD4 is a genetic determinant to systemic lupus erythematosus and involved in murine autoimmune phenotypes. Annals of the rheumatic diseases 78 , 509-518. Aloulou, A., Ali, Y.B., Bezzine, S., Gargouri, Y., and Gelb, M.H. (2012). Phospholipases: an overview. Methods in molecular biology (Clifton, NJ) 861 , 63-85. Altman, J., and Das, G.D. (1965). Autoradiographic and histological evidence of postnatal hippocampal neurogenesis in rats. The Journal of comparative neurology 124 , 319-335. Ammar, M.R., Thahouly, T., Hanauer, A., Stegner, D., Nieswandt, B., and Vitale, N. (2015). PLD1 participates in BDNF-induced signalling in cortical neurons. Scientific reports 5 , 14778. Anacker, C., and Hen, R. (2017). Adult hippocampal neurogenesis and cognitive flexibility - linking memory and mood. Nature reviews Neuroscience 18 , 335-346. Anney, R., Klei, L., Pinto, D., Regan, R., Conroy, J., Magalhaes, T.R., Correia, C., Abrahams, B.S., Sykes, N., Pagnamenta, A.T. , et al. (2010). A genome-wide scan for common alleles affecting risk for autism. Human molecular genetics 19 , 4072-4082. Bannerman, D.M., Sprengel, R., Sanderson, D.J., McHugh, S.B., Rawlins, J.N., Monyer, H., and Seeburg, P.H. (2014). Hippocampal synaptic plasticity, spatial memory and anxiety. Nature reviews Neuroscience 15 , 181-192. Bear, M.F. (1996). A synaptic basis for memory storage in the cerebral cortex. Proceedings of the National Academy of Sciences of the United States of America 93 , 13453-13459. Bienkowski, M.S., Bowman, I., Song, M.Y., Gou, L., Ard, T., Cotter, K., Zhu, M., Benavidez, N.L., Yamashita, S., Abu-Jaber, J. , et al. (2018). Integration of gene expression and brain-wide connectivity reveals the multiscale organization of mouse hippocampal networks. Nature neuroscience 21 , 1628-1643. Bourne, K.Z., Natarajan, C., Perez, C.X.M., Tumurbaatar, B., Taglialatela, G., and Krishnan, B. (2019). Suppressing aberrant phospholipase D1 signaling in 3xTg Alzheimer's disease mouse model promotes synaptic resilience. Scientific reports 9 , 18342. Bravo, F.V., Da Silva, J., Chan, R.B., Di Paolo, G., Teixeira-Castro, A., and Oliveira, T.G. (2018). Phospholipase D functional ablation has a protective effect in an Alzheimer's disease Caenorhabditis elegans model. Scientific reports 8 , 3540. Bruntz, R.C., Lindsley, C.W., and Brown, H.A. (2014). Phospholipase D signaling pathways and phosphatidic acid as therapeutic targets in cancer. Pharmacological reviews 66 , 1033-1079. 24 Bumpus, T.W., and Baskin, J.M. (2016). A Chemoenzymatic Strategy for Imaging Cellular Phosphatidic Acid Synthesis. Angewandte Chemie (International ed in English) 55 , 1315513158. Bumpus, T.W., Liang, F.J., and Baskin, J.M. (2018). Ex Uno Plura: Differential Labeling of Phospholipid Biosynthetic Pathways with a Single Bioorthogonal Alcohol. Biochemistry 57 , 226230. Burkhardt, U., Stegner, D., Hattingen, E., Beyer, S., Nieswandt, B., and Klein, J. (2014). Impaired brain development and reduced cognitive function in phospholipase D-deficient mice. Neuroscience letters 572 , 48-52. Cai, D., Netzer, W.J., Zhong, M., Lin, Y., Du, G., Frohman, M., Foster, D.A., Sisodia, S.S., Xu, H., Gorelick, F.S. , et al. (2006a). Presenilin-1 uses phospholipase D1 as a negative regulator of betaamyloid formation. Proceedings of the National Academy of Sciences of the United States of America 103 , 1941-1946. Cai, D., Zhong, M., Wang, R., Netzer, W.J., Shields, D., Zheng, H., Sisodia, S.S., Foster, D.A., Gorelick, F.S., Xu, H. , et al. (2006b). Phospholipase D1 corrects impaired betaAPP trafficking and neurite outgrowth in familial Alzheimer's disease-linked presenilin-1 mutant neurons. Proceedings of the National Academy of Sciences of the United States of America 103 , 19361940. Cazzolli, R., Shemon, A.N., Fang, M.Q., and Hughes, W.E. (2006). Phospholipid signalling through phospholipase D and phosphatidic acid. IUBMB life 58 , 457-461. Cermenati, G., Mitro, N., Audano, M., Melcangi, R.C., Crestani, M., De Fabiani, E., and Caruso, D. (2015). Lipids in the nervous system: from biochemistry and molecular biology to pathophysiology. Biochim Biophys Acta 1851 , 51-60. Checa, A., Bedia, C., and Jaumot, J. (2015). Lipidomic data analysis: tutorial, practical guidelines and applications. Analytica chimica acta 885 , 1-16. Chen, Q., Hongu, T., Sato, T., Zhang, Y., Ali, W., Cavallo, J.A., van der Velden, A., Tian, H., Di Paolo, G., Nieswandt, B. , et al. (2012). Key roles for the lipid signaling enzyme phospholipase d1 in the tumor microenvironment during tumor angiogenesis and metastasis. Science signaling 5 , ra79. Chen, W.C., Wang, W.C., Okada, Y., Chang, W.P., Chou, Y.H., Chang, H.H., Huang, J.D., Chen, D.Y., and Chang, W.C. (2017). rs2841277 (PLD4) is associated with susceptibility and rs4672495 is associated with disease activity in rheumatoid arthritis. Oncotarget 8 , 6418064190. Chen, Y.G., Siddhanta, A., Austin, C.D., Hammond, S.M., Sung, T.C., Frohman, M.A., Morris, A.J., and Shields, D. (1997). Phospholipase D stimulates release of nascent secretory vesicles from the trans-Golgi network. The Journal of cell biology 138 , 495-504. Chevaleyre, V., and Siegelbaum, S.A. (2010). Strong CA2 pyramidal neuron synapses define a powerful disynaptic cortico-hippocampal loop. Neuron 66 , 560-572. 25 Chiang, M.C., Huang, A.J.Y., Wintzer, M.E., Ohshima, T., and McHugh, T.J. (2018). A role for CA3 in social recognition memory. Behavioural brain research 354 , 22-30. Choi, S.Y., Huang, P., Jenkins, G.M., Chan, D.C., Schiller, J., and Frohman, M.A. (2006). A common lipid links Mfn-mediated mitochondrial fusion and SNARE-regulated exocytosis. Nature cell biology 8 , 1255-1262. Choi, W.S., Kim, Y.M., Combs, C., Frohman, M.A., and Beaven, M.A. (2002). Phospholipases D1 and D2 regulate different phases of exocytosis in mast cells. Journal of immunology (Baltimore, Md : 1950) 168 , 5682-5689. Citri, A., and Malenka, R.C. (2008). Synaptic plasticity: multiple forms, functions, and mechanisms. Neuropsychopharmacology : official publication of the American College of Neuropsychopharmacology 33 , 18-41. Colley, W.C., Sung, T.C., Roll, R., Jenco, J., Hammond, S.M., Altshuller, Y., Bar-Sagi, D., Morris, A.J., and Frohman, M.A. (1997). Phospholipase D2, a distinct phospholipase D isoform with novel regulatory properties that provokes cytoskeletal reorganization. Current biology : CB 7 , 191201. Corkin, S. (2002). What's new with the amnesic patient H.M.? Nature reviews Neuroscience 3 , 153-160. Corrotte, M., Chasserot-Golaz, S., Huang, P., Du, G., Ktistakis, N.T., Frohman, M.A., Vitale, N., Bader, M.F., and Grant, N.J. (2006). Dynamics and function of phospholipase D and phosphatidic acid during phagocytosis. Traffic (Copenhagen, Denmark) 7 , 365-377. Cruchaga, C., Karch, C.M., Jin, S.C., Benitez, B.A., Cai, Y., Guerreiro, R., Harari, O., Norton, J., Budde, J., Bertelsen, S. , et al. (2014). Rare coding variants in the phospholipase D3 gene confer risk for Alzheimer's disease. Nature 505 , 550-554. Dall'Armi, C., Hurtado-Lorenzo, A., Tian, H., Morel, E., Nezu, A., Chan, R.B., Yu, W.H., Robinson, K.S., Yeku, O., Small, S.A. , et al. (2010). The phospholipase D1 pathway modulates macroautophagy. Nature communications 1 , 142-142. Di Paolo, G., and Kim, T.W. (2011). Linking lipids to Alzheimer's disease: cholesterol and beyond. Nature reviews Neuroscience 12 , 284-296. Elvers, M., Stegner, D., Hagedorn, I., Kleinschnitz, C., Braun, A., Kuijpers, M.E., Boesl, M., Chen, Q., Heemskerk, J.W., Stoll, G. , et al. (2010). Impaired alpha(IIb)beta(3) integrin activation and shear-dependent thrombus formation in mice lacking phospholipase D1. Science signaling 3 , ra1. Fahy, E., Subramaniam, S., Brown, H.A., Glass, C.K., Merrill, A.H., Jr., Murphy, R.C., Raetz, C.R., Russell, D.W., Seyama, Y., Shaw, W. , et al. (2005). A comprehensive classification system for lipids. J Lipid Res 46 , 839-861. Fahy, E., Subramaniam, S., Murphy, R.C., Nishijima, M., Raetz, C.R., Shimizu, T., Spener, F., van Meer, G., Wakelam, M.J., and Dennis, E.A. (2009). Update of the LIPID MAPS comprehensive classification system for lipids. J Lipid Res 50 Suppl , S9-14. 32 and phosphatidic acid production after purinergic (P2Y6) receptor stimulation. The Journal of biological chemistry 288 , 20477-20487. Scoville, W.B., and Milner, B. (1957). Loss of recent memory after bilateral hippocampal lesions. Journal of neurology, neurosurgery, and psychiatry 20 , 11-21. Segal, M., Richter-Levin, G., and Maggio, N. (2010). Stress-induced dynamic routing of hippocampal connectivity: a hypothesis. Hippocampus 20 , 1332-1338. Selvy, P.E., Lavieri, R.R., Lindsley, C.W., and Brown, H.A. (2011). Phospholipase D: enzymology, functionality, and chemical modulation. Chemical reviews 111 , 6064-6119. Shah, S., Lubeck, E., Zhou, W., and Cai, L. (2017). seqFISH Accurately Detects Transcripts in Single Cells and Reveals Robust Spatial Organization in the Hippocampus. Neuron 94 , 752758.e751. Simons, K., and Sampaio, J.L. (2011). Membrane organization and lipid rafts. Cold Spring Harbor perspectives in biology 3 , a004697. Sonoda, H., Aoki, J., Hiramatsu, T., Ishida, M., Bandoh, K., Nagai, Y., Taguchi, R., Inoue, K., and Arai, H. (2002). A novel phosphatidic acid-selective phospholipase A1 that produces lysophosphatidic acid. The Journal of biological chemistry 277 , 34254-34263. Stace, C.L., and Ktistakis, N.T. (2006). Phosphatidic acidand phosphatidylserine-binding proteins. Biochim Biophys Acta 1761 , 913-926. Stackman, R.W., Jr., Cohen, S.J., Lora, J.C., and Rios, L.M. (2016). Temporary inactivation reveals that the CA1 region of the mouse dorsal hippocampus plays an equivalent role in the retrieval of long-term object memory and spatial memory. Neurobiology of learning and memory 133 , 118-128. Strange, B.A., Witter, M.P., Lein, E.S., and Moser, E.I. (2014). Functional organization of the hippocampal longitudinal axis. Nature reviews Neuroscience 15 , 655-669. Suh, J., Rivest, A.J., Nakashiba, T., Tominaga, T., and Tonegawa, S. (2011). Entorhinal cortex layer III input to the hippocampus is crucial for temporal association memory. Science (New York, NY) 334 , 1415-1420. Tamamaki, N., and Nojyo, Y. (1993). Projection of the entorhinal layer II neurons in the rat as revealed by intracellular pressure-injection of neurobiotin. Hippocampus 3 , 471-480. Tan, M., Li, J., Ma, F., Zhang, X., Zhao, Q., and Cao, X. (2019). PLD3 Rare Variants Identified in Late-Onset Alzheimer's Disease Affect Amyloid-beta Levels in Cellular Model. Frontiers in neuroscience 13 , 116. Tanguy, E., Costé de Bagneaux, P., Kassas, N., Ammar, M.R., Wang, Q., Haeberlé, A.M., Raherindratsara, J., Fouillen, L., Renard, P.Y., Montero-Hadjadje, M. , et al. (2020). Monoand Poly-unsaturated Phosphatidic Acid Regulate Distinct Steps of Regulated Exocytosis in Neuroendocrine Cells. Cell reports 32 , 108026. 33 Tanguy, E., Tran Nguyen, A.P., Kassas, N., Bader, M.F., Grant, N.J., and Vitale, N. (2019). Regulation of Phospholipase D by Arf6 during FcγR-Mediated Phagocytosis. Journal of immunology (Baltimore, Md : 1950) 202 , 2971-2981. Tei, R., and Baskin, J.M. (2020). Spatiotemporal control of phosphatidic acid signaling with optogenetic, engineered phospholipase Ds. The Journal of cell biology 219 . Terao, C., Ohmura, K., Kawaguchi, Y., Nishimoto, T., Kawasaki, A., Takehara, K., Furukawa, H., Kochi, Y., Ota, Y., Ikari, K. , et al. (2013). PLD4 as a novel susceptibility gene for systemic sclerosis in a Japanese population. Arthritis and rheumatism 65 , 472-480. Thakur, R., Panda, A., Coessens, E., Raj, N., Yadav, S., Balakrishnan, S., Zhang, Q., Georgiev, P., Basak, B., Pasricha, R. , et al. (2016). Phospholipase D activity couples plasma membrane endocytosis with retromer dependent recycling. eLife 5 . Thielmann, I., Stegner, D., Kraft, P., Hagedorn, I., Krohne, G., Kleinschnitz, C., Stoll, G., and Nieswandt, B. (2012). Redundant functions of phospholipases D1 and D2 in platelet alphagranule release. Journal of thrombosis and haemostasis : JTH 10 , 2361-2372. Thompson, C.L., Pathak, S.D., Jeromin, A., Ng, L.L., MacPherson, C.R., Mortrud, M.T., Cusick, A., Riley, Z.L., Sunkin, S.M., Bernard, A. , et al. (2008). Genomic anatomy of the hippocampus. Neuron 60 , 1010-1021. Trivedi, P., Kumar, R.K., Iyer, A., Boswell, S., Gerarduzzi, C., Dadhania, V.P., Herbert, Z., Joshi, N., Luyendyk, J.P., Humphreys, B.D. , et al. (2017). Targeting Phospholipase D4 Attenuates Kidney Fibrosis. Journal of the American Society of Nephrology : JASN 28 , 3579-3589. Trujillo Viera, J., El-Merahbi, R., Nieswandt, B., Stegner, D., and Sumara, G. (2016). Phospholipases D1 and D2 Suppress Appetite and Protect against Overweight. PloS one 11 , e0157607. Tsien, J.Z., Huerta, P.T., and Tonegawa, S. (1996). The essential role of hippocampal CA1 NMDA receptor-dependent synaptic plasticity in spatial memory. Cell 87 , 1327-1338. van der Lee, S.J., Holstege, H., Wong, T.H., Jakobsdottir, J., Bis, J.C., Chouraki, V., van Rooij, J.G., Grove, M.L., Smith, A.V., Amin, N. , et al. (2015). PLD3 variants in population studies. Nature 520 , E2-3. van Meer, G. (2005). Cellular lipidomics. The EMBO journal 24 , 3159-3165. van Strien, N.M., Cappaert, N.L., and Witter, M.P. (2009). The anatomy of memory: an interactive overview of the parahippocampal-hippocampal network. Nature reviews Neuroscience 10 , 272-282. Vattulainen, I., and Rog, T. (2011). Lipid simulations: a perspective on lipids in action. Cold Spring Harbor perspectives in biology 3 . Vermeren, M.M., Zhang, Q., Smethurst, E., Segonds-Pichon, A., Schrewe, H., and Wakelam, M.J. (2016). The Phospholipase D2 Knock Out Mouse Has Ectopic Purkinje Cells and Suffers from Early Adult-Onset Anosmia. PloS one 11 , e0162814. 34 Vitale, N., Caumont, A.S., Chasserot-Golaz, S., Du, G., Wu, S., Sciorra, V.A., Morris, A.J., Frohman, M.A., and Bader, M.F. (2001). Phospholipase D1: a key factor for the exocytotic machinery in neuroendocrine cells. The EMBO journal 20 , 2424-2434. Voigt, F., Reuter, M., Kasaruho, A., Schulz, E.C., Pillai, R.S., and Barabas, O. (2012). Crystal structure of the primary piRNA biogenesis factor Zucchini reveals similarity to the bacterial PLD endonuclease Nuc. RNA (New York, NY) 18 , 2128-2134. Wang, Z., Zhang, F., He, J., Wu, P., Tay, L.W.R., Cai, M., Nian, W., Weng, Y., Qin, L., Chang, J.T. , et al. (2017). Binding of PLD2-Generated Phosphatidic Acid to KIF5B Promotes MT1-MMP Surface Trafficking and Lung Metastasis of Mouse Breast Cancer Cells. Developmental cell 43 , 186-197.e187. Wenk, M.R. (2005). The emerging field of lipidomics. Nature reviews Drug discovery 4 , 594-610. Yao, Y., Wang, X., Li, H., Fan, J., Qian, X., Li, H., and Xu, Y. (2020). Phospholipase D as a key modulator of cancer progression. Biological reviews of the Cambridge Philosophical Society. Yoshikawa, F., Banno, Y., Otani, Y., Yamaguchi, Y., Nagakura-Takagi, Y., Morita, N., Sato, Y., Saruta, C., Nishibe, H., Sadakata, T. , et al. (2010). Phospholipase D family member 4, a transmembrane glycoprotein with no phospholipase D activity, expression in spleen and early postnatal microglia. PloS one 5 , e13932. Zhang, Y., Zhu, H., Zhang, Q., Li, M., Yan, M., Wang, R., Wang, L., Welti, R., Zhang, W., and Wang, X. (2009). Phospholipase dalpha1 and phosphatidic acid regulate NADPH oxidase activity and production of reactive oxygen species in ABA-mediated stomatal closure in Arabidopsis. The Plant cell 21 , 2357-2377. Zucker, R.S., and Regehr, W.G. (2002). Short-term synaptic plasticity. Annual review of physiology 64 , 355-405. 35 CHAPTER 2 Phospholipase D1 Ablation Disrupts Mouse Longitudinal Hippocampal Axis Organization and Functioning 36 37 38 39 40 41 48 49 50 51 52 53 54 55 CHAPTER 2 – Supplementary Information Phospholipase D1 Ablation Disrupts Mouse Longitudinal Hippocampal Axis Organization and Functioning 56 57 64 CHAPTER 3 Phospholipase D2 ablation leads to deficits in social memory (Manuscript to be submitted) Luísa Santa-Marinha,1,2 Francisca Vaz Bravo,1,2 André Miguel Miranda1,2, Torcato Meira,1,2 Vítor Pinto,1,2 and Tiago Gil Oliveira 1,2 1Life and Health Sciences Research Institute (ICVS), School of Medicine, University of Minho, Campus Gualtar, 4710-057 Braga, Portugal 2ICVS/3B’s - PT Government Associate Laboratory, Braga/Guimarães, Portugal 65 Abstract The hippocampus is a temporal brain region crucial for learning and memory organized along a longitudinal dorsal-ventral axis in mice. Classically, the dorsal hippocampus (DH) is preferentially associated with spatial navigation and episodic memory and the ventral hippocampus (VH) with emotional stress-related behaviors. Its cross-sectional architecture is constituted by the dentate gyrus and Cornu Ammonis subfields, CA1, CA2 and CA3. Previous work showed a decreasing phosphatidic acid (PA) gradient along the longitudinal axis of the rodent hippocampus, suggesting the phospholipase D (PLD) pathway could be involved in DH-VH axis regulation. Even though the PLD superfamily includes six members (PLD1-PLD6), only two of them, PLD1 and PLD2, are shown to hydrolyze phosphatidylcholine into PA. We previously showed PLD1 as a major contributor for total PA production in the mouse hippocampus compared to PLD2, and that PLD1 ablation leads to behavioral deficits in novelty recognition and social interaction, to deficits in long-term depression and changes in specific synaptic protein in the DH. Here, we expand the observations on the role of the PLD pathway in DH-VH organization and functioning. We show that PLD2 ablation leads to littermate recognition impairment and decreases social exploration in a CA2-dependent task. We also performed a complete electrophysiological characterization studying the effect of PLD2 ablation in the DH and VH, and we observed that it specifically reduces long-term potentiation (LTP) in the VH CA1. Finally, we found no major impact on synaptic protein levels upon ablation of PLD2 in either the DH or VH. Since PLD2 is highly expressed in CA2, and since it was previously shown that CA2 impacts social memory in a circuit that depends on VH CA1, our results suggest a potential role for PLD2 in “dorsal CA2” – “ventral CA1” circuit regulation with an impact in social memory recall, and potential implications for the pathophysiology of neuropsychiatric disorders. 66 Introduction The hippocampus is a temporal brain region crucial for learning and memory. Its transversal organization is preserved along the longitudinal axis and is constituted by the dentate gyrus (DG) and Cornu Ammonis (CA) subfields, consisting of CA1, CA2 and CA3 (Strange et al., 2014). Despite this intrinsic circuitry being preserved throughout the longitudinal axis and across species, anatomical features (such as cortical and subcortical connections), electrophysiological, gene expression and functional studies segregate a longitudinal dorsal-ventral (DH-VH) axis in rodents, which correspond in primates to posterior-anterior poles (Strange et al., 2014). Classically, the DH is preferentially associated with spatial navigation and episodic memory and the VH with emotional stress-related behaviors (Bannerman et al., 2014; Fanselow and Dong, 2010; Kheirbek et al., 2013; McHugh et al., 2011; Strange et al., 2014) although a gradual biochemical and functional transition between the two poles has been approached (Bienkowski et al., 2018; Maggio and Segal, 2007; Miranda et al., 2019; Pinto et al., 2015; Shah et al., 2017; Thompson et al., 2008). The importance of hippocampal subregions has been extensively addressed by inhibition or genetic manipulations of these structures. CA1 has been shown to be necessary for spatial memory (Tsien et al., 1996). Specifically, dorsal CA1 was shown to be involved in contextual memory, remote memory recall and spatial and object memory (Goshen et al., 2011; Roy et al., 2017; Stackman et al., 2016) and ventral CA1 (and its projections to the nucleus accumbens) was implicated in the storage of social memory (Okuyama et al., 2016). CA3 is important for rapid one-trial contextual learning and for pattern completion recall (Nakashiba et al., 2008) and for social memory (Chiang et al., 2018). Regarding the DG, where neurogenesis persists into adulthood, it was shown that pattern separation depends on adult-born young granule cells, while recall by pattern completion relies on older granule cells (Nakashiba et al., 2012). CA2 is a morphological, molecular and electrophysiological region with distinct synaptic connectivity and gene expression profile (Dudek et al., 2016). Importantly, genetically targeted inactivation of dorsal CA2 pyramidal neurons causes lack of social memory (ability of an animal to remember a conspecific) but no alterations in sociability or other hippocampus dependent behaviors (Hitti and Siegelbaum, 2014). A recent study showed that dorsal CA2 plays an active role in social memory encoding, consolidation, and recall and that dorsal CA2 projections to ventral CA1 are necessary for social memory (Meira et al., 2018). 67 Glycerophospholipids can be remodeled in order to generate products that can serve as second messengers by different classes of phospholipases that act on different positions of the phosphodiester bond (Panda et al., 2018). In mammals, the phospholipase D (PLD) superfamily includes six members (PLD1-PLD6), but only two of them, PLD1 and PLD2, are able to hydrolyze the membrane phospholipid phosphatidylcholine (PC) into the metabolically active phosphatidic acid (PA) (Frohman, 2015; Oliveira et al., 2010; Yao et al., 2020). Curiously, there is a distinctive lipid composition along the hippocampal longitudinal axis, mainly in the relative abundance of sphingolipids and phospholipids. Particularly, the DH presents increased PA and decreased PC when compared to VH, potentially implicating the PLD pathway in DH-VH axis regulation (Miranda et al., 2019). Previous studies showed that PLD1 and PLD2 are the only contributors to total PLD activity in the mouse brain, although PLD1 is a major source of PA in both rodent DH and VH when compared to PLD2 (see chapter 2) (Santa-Marinha et al., 2020). PLD1 ablation affects predominantly the lipidome of the DH and Pld1 knockout (KO) mice present specific deficits in novel object recognition and social interaction, disruption in the DH-VH dendritic arborization differentiation in CA1/CA3 pyramidal neurons, reduced long-term depression (LTD) induction and reduced GluN2A and SNAP-25 protein levels in the DH. Here we expand the observations on the role of PLD1 in DH-VH organization and show that PLD2 ablation leads to social discrimination deficits, namely littermate recognition impairment, decreases social exploration and reduces long-term potentiation (LTP) in the VH, showing that, although PLD1 and PLD2 catalyze the same reaction, their specific ablation promotes different phenotypes. 68 Materials and Methods Animals Pld2 -/- mice were generated through the elimination of exons 13-15, including exon 14 (that encodes for the first HKD motif of PLD2), using a Cre-lox/FLP-FRT recombination system, as previously described (Oliveira et al., 2010). Pld2 +/- mice (C57BL/6 background) were crossed so littermate mice could be used for the procedures. All experimental procedures were performed in agreement with the Portuguese authority for animal experimentation, Direção Geral de Alimentação e Veterinária (DGAV), the Directive 2010/63/EU guidelines and approved by the local committee. All mice had ad libitum access to water and food and were maintained on a 12h light/dark cycle. All the procedures were performed during daytime, except for the social discrimination, sociability and preference for social novelty behavioral tests. Prior to all behavioral tests, animals were placed in the testing room for one hour in order to acclimatize. All apparatus were cleaned with 10% ethanol solution between testing subjects. Animal Behavior Fear Conditioning (FC) The Fear Conditioning (FC) protocol aimed to test contextual and cued memory. 3.5-7.5 months male mice were tested. Standard operant chambers (Med Associates Inc.) (22 x 19 x 12.5 cm) with a grid on the bottom where shocks were transmitted was used, except on day 3. On the first day mice were placed in the arena and allowed to explore for 5 minutes. Then a tone (80 dB) was presented for 30 seconds and in the last second a mild shock (0.5 mA for 1 second) was given for fear acquisition. This was repeated for 5 blocks, with an interval of 30 seconds between then. To evaluate the contextual fear conditioning on the second day, mice were only placed in the arena and allowed to explore for 5 minutes. On the third day mice were placed in a different arena (24 x 32 x 18.5 cm) and allowed to explore for 5 minutes. Then 5 blocks of 30 seconds of tone and 30 seconds of interval were given in order to evaluate cued fear conditioning. For the next 5 days mice were placed in the arena and give 15 blocks of 30 seconds of tone followed by 30 seconds of interval, to evaluate the extinction of the freezing response. 30 days after the last day of extinction protocol, mice were placed in the arena and allowed to explore for 5 minutes. Then 5 blocks of 30 seconds of tone followed by 30 seconds of interval were presented to 69 evaluate spontaneous recovery. Animals were video-recorded and the percentage of freezing behavior was analyzed. All stimulation procedures were done through Med-PC IV (Med Associates Inc.) software, except for the shock that was manually given. Three-chamber Social Discrimination and Three-chamber Sociability and Preference for Social Novelty These tests were adapted from previously described protocols (Meira et al., 2018; Moy et al., 2007). 3-6 months male mice were used for the social tests. A transparent rectangular arena (56 cm long x 50 cm wide x 39 cm high) was divided in three equal chambers with openings that allowed access into each chamber. Trials consisted of 10-minute period each, where the subject mice were allowed to explore the arena. When the trial ended and the animal stopped in the center arena, the access to the other chambers was blocked with two doors to switch trials. The social discrimination test consisted of three trials. In the first habituation trial, mice were individually placed in the center of the arena and freely explore. In the second habituation trial, two empty round wire cups (9.8 cm high, with a bottom diameter of 8 cm and several horizontal opened bars spaced 3.7 x 0.5 cm apart which allowed nose contact between the bars) were placed on the side chambers and the subject mice freely explored the arena. In the third trial, a co-housed littermate and an unfamiliar male stranger with no prior contact with the tested mouse were each placed inside one of the wire cups. The location of the stranger and the littermate mouse was systematically alternated between the tested mice. The sociability and preference for social novelty test consisted of four trials. The first and second trials were equal to the ones previously described. In the third trial, an unfamiliar male mice (stranger 1), which had no prior contact with the subject mice, was placed inside one of the cups to test sociability of the subject mice. In the fourth trial, a second stranger male mice (stranger 2) was enclosed in the previously empty cup and preference for social novelty was assessed. The location of the strangers was systematically alternated between the tested mice. Behavior was video-recorded and the amount of time spent interacting with each cup was manually scored. Social discrimination, social preference and preference for social novelty scores were calculated as the difference between the time spent exploring the cups placed on the side chambers, divided by the total time exploring both. Testing was conducted under red light. 70 Electrophysiology Electrophysiological recordings were performed according to a protocol described elsewhere (Pinto et al., 2015). Slices from 2-3 months male and female mice were used. Briefly, after intraperitoneal injection of Na+-pentobarbital (40mg/kg) anesthesia and decapitation, the brain was rapidly removed and placed in ice-cold sucrose-based solution containing the following (in mM): 2.5 KCl, 7 MgCl2, 1.25 NaH2PO4, 110 sucrose, 26 NaHCO3, 10 glucose, bubbled with carbogen gas (95% O2, 5% CO2). Slices of 300 μm were cut transverse to the axis of the hippocampus using a tissue slicer (Leica VT 1200s) and placed in a container with artificial cerebrospinal fluid (ACSF) containing (in mM): 124 NaCl, 2.5 KCl, 1 MgSO4, 2 CaCl2, 1.25 NaH2PO4, 26 NaHCO3, 10 glucose, bubbled with carbogen gas and maintained at 33ºC for 30 minutes. Before recording, the slices were then stored at room temperature for a minimum of 30 minutes. Extracellular recordings were performed in a submerge-type chamber bathed with ACSF at 31ºC. A custom-made bipolar tungsten stimulation electrode was placed in the transition between CA1 and CA3, at the Schaffer collaterals, and a recording borosilicate glass pipette filled with ACSF (3-5 MΩ) was positioned in the middle of the stratum radiatum of CA1 at a predetermined fixed distance of approximately 1mm apart the stimulation electrode. Recordings were digitized with a Multiclamp 700B amplifier (Axon Instruments) Signals were low-pass filtered at 3 kHz and sampled at 10 kHz. Stimulation strength was adjusted to 30–50 % of the maximum field excitatory post synaptic potential (fEPSP) slope, after a fixed protocol of 4 increasing stimulus (2000-8000mV) was applied (input-output relationship). Before baseline acquisition, a series of paired pulse stimuli of varying interpulse interval (25, 50, 100 and 300ms) were given. The paired pulse (PP) ratio was calculated by dividing the slope of fEPSP2 by fEPSP1. A minimum of 10 minute stable baseline recordings were acquired at 0.03 Hz. LTD was elicited by delivering a low-frequency stimulation protocol consisting of 15 min 1 Hz stimulation. LTP was elicited by delivering 3 θ-burst stimuli (θ-burst: 100 Hz burst of four pulses repeated at 5 Hz with each tetanus including 10-burst trains) separated by 15 s intervals. All stored curves were an average of four consecutive recordings. Maximum EPSP slopes were calculated off-line using the WinLTP software. For LTP and LTD analysis, all individual slopes were normalized to the average slope of baseline recordings. 71 Western Blot After cervical dislocation and hippocampi dissection, they were divided in approximately three equal dorsal, intermediate and ventral parts. Brains from 3.5-7 months male and female mice were used. Samples were diluted in radioimmunoprecipitation assay buffer (RIPA) containing 150 nM NaCl, 1.0% triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM TrisBase pH=8 and supplemented with protease (Roche) and phosphatase inhibitor cocktails (Sigma-Aldrich). After homogenization, samples were mixed in a rotator at 4ºC for 2 hours at 40rpm and then centrifuged at 4ºC for 20 minutes at 14000rpm. Supernatants were quantified (BCA, Pierce) and diluted to equal concentration in RIPA. Samples were prepared with NuPAGE LDS sample buffer and NuPAGE reducing reagent and loaded in NuPAGE 4–12% Bis–Tris gels. MES SDS running buffer (NuPAGE) was used for separation. Wet transfer used 50% running buffer, 20% methanol and 30% deionized water and was made on 0.45µm nitrocellulose membranes (Amersham) for 2 hours at 100V. Membrane blockage was performed with 5% dried milk (Nestle) for 1 hour at room temperature. Incubation with primary antibodies, diluted in 2.5% dried milk or bovine serum albumin (BSA), was performed overnight at 4ºC and incubation with HRP-conjugated secondary antibodies, diluted in 2.5% dried milk or BSA, was performed for 1 hour at room temperature. Chemiluminescence signal was detected with ChemiDoc XRS+ (Bio-Rad) and membrane development was achieved with Clarity Western ECL Substrate (Bio-Rad) or SuperSignal West Femto Maximum Sensitivity Substrate (ThermoScientific). ImageJ software was used for quantification. Primary antibodies used were: α-Tubulin (T6074, Sigma-Aldrich, 1:10000); GAPDH (Cell Signaling Technology, 1:3000); Synaptophysin (Synaptic Systems, 1:5000); SNAP-25 (Abcam, 1:5000); PSD95 (Abcam, 1:5000); Homer (Abcam, 1:3000); GluA1/GluR1 (Millipore, 1:1000); GluA2/GluR2 (Abcam, 1:1000); GluN2A/NR2A (Millipore, 1:1000); GluN2B/NR2B (Abcam, 1:1000) and pY1472-GluN2B/NR2B (Abcam, 1:1000). HRPconjugated secondary antibodies used were: Goat Anti-rabbit IgG (BioRad) and Goat Anti-mouse IgG (BioRad). Quantification and Statistical Analysis Western blot analysis was performed using ImageJ (NIH). The statistical analysis was performed using GraphPad Prism 7.00 software. Statistical significance was assessed by two-way ANOVA and Student’s t test, and referenced in figure legends whenever used. Values were accepted as 72 significant when p<0.05. *p<0.05, **p< 0.01, ***p<0.001. Number of mice for all statistical analyses is indicated in each legend. Values are expressed as mean±SEM. 73 Results PLD2 ablation impairs social discrimination and decreases social exploration in a hippocampusdependent social discrimination task In order to evaluate the impact of PLD2 ablation on hippocampal functioning, we performed hippocampal-associated behaviors. First, when performing the FC test, we observe that Pld2 KO animals had similar freezing responses when comparing to wild-type (WT) mice when contextual and cued memories were evaluated (Figure 1A, left panel), and in the 5 subsequent days in the extinction freezing upon a cue protocol (Figure 1A, middle panel). Curiously, 30 days later, KO animals tend to freeze less when exposed to the context and cue (Figure 1A, right panel). Then, we performed social tests, in order to evaluate social preference and preference for social novelty and social discrimination. The ablation of PLD2 does not impair the social preference (Figure 1B and S1A) neither the preference for social novelty (Figure 1C and S1B) but impairs social discrimination by reducing the ability to recognize a littermate (Figure 1D, left panel, and S1C). Moreover, Pld2 KO mice were less social in a hippocampus-dependent task, considering that they spent less time exploring (Figure 1D, right panel). 80 to varying degrees, depending on neuron types. Within the hippocampus, CA2 has noticeably more signal than CA1 and CA3 regions, which, in turn, express far more than the DG, where PLD2 is almost undetected (Vermeren et al., 2016). Moreover, dorsal CA2 provides an excitatory input to the same ventral CA1 subregion that projects to the nucleus accumbens shell, implicated in social memory (Meira et al., 2018). Curiously, mice lacking PLD2, highly expressed in CA2, have reduced LTP when Schaffer collaterals are stimulated in VH (see figure 2A, right panel). Based on these behavioral observations, one possibility is that PLD2 specifically affects the dorsal CA2 – ventral CA1 circuit, which could explain the connection with social memory. CA2 region has features that distinguish it from CA1 and CA3, from synaptic connectivity and electrophysiological characteristics to gene expression profile and relative resistance to cell death (Dudek et al., 2016). CA2 has high resistance to damage from injury (Nadler et al., 1978; Sloviter and Damiano, 1981) and resistance to synaptic plasticity, namely failure to display LTP and LTD compared with other CA regions (Zhao et al., 2007). Several classes of proteins are particularly enriched in CA2, such as calcium regulators, extracellular matrix components, proteins involved in growth factorand G protein-coupled receptor (GPCR)-mediated signaling, and intracellular signal transduction proteins (Dudek et al., 2016). Arginine vasopressin (AVP) receptor 1b (AVPR1b) and oxytocin (OXT) receptor (OXTR) are also highly expressed in CA2 pyramidal neurons (Hammock and Levitt, 2013; Hidema et al., 2016; Mitre et al., 2016; Yoshida et al., 2009; Young et al., 2006). OXT and AVP are two hypothalamic produced neuropeptides. OXT is first known as a regulator of parturition and lactation and AVP is a regulator of extracellular fluid volume and a vasoconstrictor, but they have also been study for their ability to modulate social behaviors. Decreased levels of OXT and OXTR, either in full KO (Takayanagi et al., 2005) or conditional KO mice, lead to social deficits and poor social recognition (Lee et al., 2008). Recent studies showed that OXTR in the anterior DG and anterior CA2/CA3 are essential for discrimination of social stimuli (and not non-social) (Raam et al., 2017) and for long-term social recognition memory, without any effect on sociability and preference for social novelty (Lin et al., 2018). Regarding AVPR1b, it was shown that AVPR1b KO mice have impairment of sociability (DeVito et al., 2009) and social memory (DeVito et al., 2009; Wersinger et al., 2002; Wersinger et al., 2008). Partial replacement of this receptor through lentiviral delivery into the dorsal CA2 is able to restore the probability of socially motivated attack behavior in AVPR1b KO mice (Pagani et al., 2015) and optogenetic release of AVP in CA2 enhances social memory encoding, but not social recall (Smith et al., 2016). 81 Association between PLD and AVP/OXT has already been described. In cultured rat glomerular mesangial cells, AVP increases the phosphatidylethanol (PEtOH) formation (indicating PLD activity) and inhibition of PLD decreases AVP produced PEtOH (Kusaka et al., 1996). Other studies implicate PLD activation by AVP in myogenesis (Komati et al., 2005; Naro et al., 1997) and PLD in AVP-stimulated Ca2+ spiking in vascular smooth muscle cells (Li et al., 2001). In fact, PLD activation by AVP in myogenic cells increases exocytosis and vesicle traffic (Coletti et al., 2000). PLD activity in cultured amnion cells is activated by the addition of AVP and OXT (Inamori et al., 1995) and OXT also activates PLD in cultured human pregnant myometrial cells (Morrison et al., 1996). We can speculate that the connections between PLD2 and OXT/AVP are dysregulated in Pld2 KO animals, thus leading to social deficits. Another interesting feature is that OXT transiently reduces synaptic inhibition in multiple brain regions and enables long-term synaptic plasticity in the auditory cortex (Mitre et al., 2016) and selective activation of AVPR1b or OXTR agonists induces significant potentiation in excitatory synaptic responses in CA2, despite its resistance to LTP (Pagani et al., 2015). This enhances the possibility that PLD2 could be responsible for synaptic electrophysiological responses with impact on social behavior, since its ablation causes deficits in LTP (see Figure2, right panel). Another possibility is that TWIK-related K+ channel (TREK), a subfamily of K2P channels lipidregulated membrane proteins, could be involved in PLD2 ablation associated phenotypes. TREK channels include TREK1, TREK2, and TWIK-related arachidonic acid stimulated K+ channel (TRAAK) (Comoglio et al., 2014). Importantly, Comoglio et al. showed that TREK1 and TREK2 are potentiated by PLD2 and that none of these channels is modulated by PLD1, since PLD2, but not PLD1, directly binds to the C terminus of TREK1 and TREK2 (and not to TRAAK). Coexpression of TREK1/TREK2 and PLD2 increases TREK1/TREK2 current (Comoglio et al., 2014). This selective binding allows a local PA production that tonically activates the channel and contributes to resting membrane potential (Comoglio et al., 2014). Remarkably, TREK1, is also highly expressed in CA2 (Talley et al., 2001) and TREK1 ablation in mice leads to decreased LTP in CA1 region, similarly to the alterations seen in the VH of Pld2 KO mice (Wang et al., 2020). Human 22q11.2 deletion syndrome (22q11.2DS) mouse model (Df(16)A+/−), also known as DiGeorge syndrome (the greatest molecular genetic risk factor for schizophrenia) display impaired social memory, that phenocopies a silent CA2 (Piskorowski et al., 2016). These mice have intrinsic decreased action potential in CA2 pyramidal neurons, with a hyperpolarized resting membrane potential. This hyperpolarization relates to the upregulated current of TREK1 channel 82 in CA2 (Piskorowski et al., 2016). One could hypothesize that the ablation of PLD2 could have an impact in the regulation of TREK1, leading to social deficits in these animals. Of note, although social deficits were already described in mice lacking PLD2 (Burkhardt et al., 2014), our careful Pld2 KO studies did not reveal impaired preference for social novelty. In agreement to previous work, PLD2 ablation did not impact the percentage of freezing in contextual FC (Figure 1A, left panel) (Oliveira et al., 2010). Pld2 KO also performed as WT in cued fear conditioning (Figure 1A, left panel) and in the extinction protocol (Figure 1A, middle panel). Despite this, there was a tendency for KO animals to freeze less than WT animals in spontaneous recovery 30 days after FC protocol (Figure 1A, tight panel). There is a great amount of evidence involving the VH in acquisition of conditioned fear, many of them focusing on the reciprocal connections of VH with the amygdala (Bast et al., 2001; Chaaya et al., 2018; Cox et al., 2013; Hobin et al., 2006; Izquierdo et al., 2016; Silva et al., 2019; Zhang et al., 2001). Interestingly, a study used a foot shock associated with optogenetic stimulation of auditory inputs targeting the amygdala for animal conditioning and then delivered optogenetical LTD and LTP conditioning to the auditory input (Nabavi et al., 2014). Curiously, LTD inactivates and LTP reactivates the memory of the shock (Nabavi et al., 2014). Since Pld2 KO mice lack the ability to induce LTP, this impairment might justify the tendency to freeze less when recalling the contextual fear memory. PLD2 has a documented role in the pathophysiology of Alzheimer’s disease (AD). In primary cortical neurons, Aβ oligomers induce total PLD activity, which is abolished in mouse cultured neurons without PLD2 (Oliveira et al., 2010). Moreover, genetic ablation of PLD2 leads to attenuation of AD pathogenesis by increasing resistance to Aβ oligomer insult. Behavioral tests with SwAPP mouse model showed that ablating PLD2 improves learning in memory in contextual FC and radial arm water maze test, thus leading to memory deficit protection (Oliveira et al., 2010). The hippocampus is an early target of AD pathology and CA2 is highly affected. Immunohistochemistry of human AD brain revealed a decrease in parvalbumin interneuron density in CA2, similar to what is found in CA1 and DG (Brady and Mufson, 1997), in agreement with findings in mouse models (Cattaud et al., 2018). The record of the activity of hippocampal place cells in Tg-F344 AD model (with human genetic mutations APPSwe and PS1ΔE9) showed that spiking activity of place cells in the CA2 and CA3 pyramidal regions in AD rats have sharply reduced spatial fidelity (Galloway et al., 2018). 83 With this work we expose PLD2 as essential for social behavior and hippocampal synaptic potentiation, enhancing a possible dysregulation of AVPR1b/OXT/TREK1 and of the connectivity from dorsal CA2 to ventral CA1, whose impairment also leads to social deficits and potentiation in excitatory synaptic. Further studies should tackle this relationship, either by colocalization studies, protein quantification or acute inhibition of PLD2. It is also mandatory to tackle the possible role of PLD2 in the pathophysiology of AD and the implications on CA2 region. 84 References Bannerman, D.M., Sprengel, R., Sanderson, D.J., McHugh, S.B., Rawlins, J.N., Monyer, H., and Seeburg, P.H. (2014). Hippocampal synaptic plasticity, spatial memory and anxiety. Nature reviews Neuroscience 15 , 181-192. Bast, T., Zhang, W.N., and Feldon, J. (2001). The ventral hippocampus and fear conditioning in rats. Different anterograde amnesias of fear after tetrodotoxin inactivation and infusion of the GABA(A) agonist muscimol. Experimental brain research 139 , 39-52. Bienkowski, M.S., Bowman, I., Song, M.Y., Gou, L., Ard, T., Cotter, K., Zhu, M., Benavidez, N.L., Yamashita, S., Abu-Jaber, J. , et al. (2018). Integration of gene expression and brain-wide connectivity reveals the multiscale organization of mouse hippocampal networks. Nature neuroscience 21 , 1628-1643. Brady, D.R., and Mufson, E.J. (1997). Parvalbumin-immunoreactive neurons in the hippocampal formation of Alzheimer's diseased brain. Neuroscience 80 , 1113-1125. Burkhardt, U., Stegner, D., Hattingen, E., Beyer, S., Nieswandt, B., and Klein, J. (2014). Impaired brain development and reduced cognitive function in phospholipase D-deficient mice. Neuroscience letters 572 , 48-52. Cattaud, V., Bezzina, C., Rey, C.C., Lejards, C., Dahan, L., and Verret, L. (2018). Early disruption of parvalbumin expression and perineuronal nets in the hippocampus of the Tg2576 mouse model of Alzheimer's disease can be rescued by enriched environment. Neurobiology of aging 72 , 147-158. Chaaya, N., Battle, A.R., and Johnson, L.R. (2018). An update on contextual fear memory mechanisms: Transition between Amygdala and Hippocampus. Neuroscience and biobehavioral reviews 92 , 43-54. Chiang, M.C., Huang, A.J.Y., Wintzer, M.E., Ohshima, T., and McHugh, T.J. (2018). A role for CA3 in social recognition memory. Behavioural brain research 354 , 22-30. Coletti, D., Silvestroni, L., Naro, F., Molinaro, M., Adamo, S., and Palleschi, S. (2000). Vesiclemediated phosphatidylcholine reapposition to the plasma membrane following hormone-induced phospholipase D activation. Experimental cell research 256 , 94-104. Comoglio, Y., Levitz, J., Kienzler, M.A., Lesage, F., Isacoff, E.Y., and Sandoz, G. (2014). Phospholipase D2 specifically regulates TREK potassium channels via direct interaction and local production of phosphatidic acid. Proceedings of the National Academy of Sciences of the United States of America 111 , 13547-13552. Cox, D., Czerniawski, J., Ree, F., and Otto, T. (2013). Time course of dorsal and ventral hippocampal involvement in the expression of trace fear conditioning. Neurobiology of learning and memory 106 , 316-323. DeVito, L.M., Konigsberg, R., Lykken, C., Sauvage, M., Young, W.S., 3rd, and Eichenbaum, H. (2009). Vasopressin 1b receptor knock-out impairs memory for temporal order. The Journal of neuroscience : the official journal of the Society for Neuroscience 29 , 2676-2683. 85 Dudek, S.M., Alexander, G.M., and Farris, S. (2016). Rediscovering area CA2: unique properties and functions. Nature reviews Neuroscience 17 , 89-102. Fanselow, M.S., and Dong, H.W. (2010). Are the dorsal and ventral hippocampus functionally distinct structures? Neuron 65 , 7-19. Frohman, M.A. (2015). The phospholipase D superfamily as therapeutic targets. Trends Pharmacol Sci 36 , 137-144. Galloway, C.R., Ravipati, K., Singh, S., Lebois, E.P., Cohen, R.M., Levey, A.I., and Manns, J.R. (2018). Hippocampal place cell dysfunction and the effects of muscarinic M(1) receptor agonism in a rat model of Alzheimer's disease. Hippocampus 28 , 568-585. Goshen, I., Brodsky, M., Prakash, R., Wallace, J., Gradinaru, V., Ramakrishnan, C., and Deisseroth, K. (2011). Dynamics of retrieval strategies for remote memories. Cell 147 , 678-689. Hammock, E.A., and Levitt, P. (2013). Oxytocin receptor ligand binding in embryonic tissue and postnatal brain development of the C57BL/6J mouse. Frontiers in behavioral neuroscience 7 , 195. Hidema, S., Fukuda, T., Hiraoka, Y., Mizukami, H., Hayashi, R., Otsuka, A., Suzuki, S., Miyazaki, S., and Nishimori, K. (2016). Generation of Oxtr cDNA(HA)-Ires-Cre Mice for Gene Expression in an Oxytocin Receptor Specific Manner. Journal of cellular biochemistry 117 , 1099-1111. Hitti, F.L., and Siegelbaum, S.A. (2014). The hippocampal CA2 region is essential for social memory. Nature 508 , 88-92. Hobin, J.A., Ji, J., and Maren, S. (2006). Ventral hippocampal muscimol disrupts context-specific fear memory retrieval after extinction in rats. Hippocampus 16 , 174-182. Inamori, K., Sagawa, N., Hasegawa, M., Itoh, H., Yano, J., and Mori, T. (1995). Activation of phospholipase D in cultured human amnion cells. Reproduction, fertility, and development 7 , 1591-1594. Izquierdo, I., Furini, C.R., and Myskiw, J.C. (2016). Fear Memory. Physiological reviews 96 , 695750. Kheirbek, M.A., Drew, L.J., Burghardt, N.S., Costantini, D.O., Tannenholz, L., Ahmari, S.E., Zeng, H., Fenton, A.A., and Hen, R. (2013). Differential control of learning and anxiety along the dorsoventral axis of the dentate gyrus. Neuron 77 , 955-968. Komati, H., Naro, F., Mebarek, S., De Arcangelis, V., Adamo, S., Lagarde, M., Prigent, A.F., and Némoz, G. (2005). Phospholipase D is involved in myogenic differentiation through remodeling of actin cytoskeleton. Molecular biology of the cell 16 , 1232-1244. Kusaka, I., Ishikawa, S.E., Higashiyama, M., Saito, T., Nagasaka, S., and Saito, T. (1996). The activation of phospholipase D participates in the mitogenic action of arginine vasopressin in cultured rat glomerular mesangial cells. Endocrinology 137 , 5421-5428. 86 Lee, H.J., Caldwell, H.K., Macbeth, A.H., Tolu, S.G., and Young, W.S., 3rd (2008). A conditional knockout mouse line of the oxytocin receptor. Endocrinology 149 , 3256-3263. Li, Y., Shiels, A.J., Maszak, G., and Byron, K.L. (2001). Vasopressin-stimulated Ca2+ spiking in vascular smooth muscle cells involves phospholipase D. American journal of physiology Heart and circulatory physiology 280 , H2658-2664. Lin, Y.T., Hsieh, T.Y., Tsai, T.C., Chen, C.C., Huang, C.C., and Hsu, K.S. (2018). Conditional Deletion of Hippocampal CA2/CA3a Oxytocin Receptors Impairs the Persistence of Long-Term Social Recognition Memory in Mice. The Journal of neuroscience : the official journal of the Society for Neuroscience 38 , 1218-1231. Maggio, N., and Segal, M. (2007). Striking variations in corticosteroid modulation of long-term potentiation along the septotemporal axis of the hippocampus. The Journal of neuroscience : the official journal of the Society for Neuroscience 27 , 5757-5765. McHugh, S.B., Fillenz, M., Lowry, J.P., Rawlins, J.N., and Bannerman, D.M. (2011). Brain tissue oxygen amperometry in behaving rats demonstrates functional dissociation of dorsal and ventral hippocampus during spatial processing and anxiety. The European journal of neuroscience 33 , 322-337. Meira, T., Leroy, F., Buss, E.W., Oliva, A., Park, J., and Siegelbaum, S.A. (2018). A hippocampal circuit linking dorsal CA2 to ventral CA1 critical for social memory dynamics. Nature communications 9 , 4163. Miranda, A.M., Bravo, F.V., Chan, R.B., Sousa, N., Di Paolo, G., and Oliveira, T.G. (2019). Differential lipid composition and regulation along the hippocampal longitudinal axis. Transl Psychiatry 9 , 144. Mitre, M., Marlin, B.J., Schiavo, J.K., Morina, E., Norden, S.E., Hackett, T.A., Aoki, C.J., Chao, M.V., and Froemke, R.C. (2016). A Distributed Network for Social Cognition Enriched for Oxytocin Receptors. The Journal of neuroscience : the official journal of the Society for Neuroscience 36 , 2517-2535. Morrison, J.J., Dearn, S.R., Smith, S.K., and Ahmed, A. (1996). Activation of protein kinase C is required for oxytocin-induced contractility in human pregnant myometrium. Human reproduction (Oxford, England) 11 , 2285-2290. Moy, S.S., Nadler, J.J., Young, N.B., Perez, A., Holloway, L.P., Barbaro, R.P., Barbaro, J.R., Wilson, L.M., Threadgill, D.W., Lauder, J.M. , et al. (2007). Mouse behavioral tasks relevant to autism: phenotypes of 10 inbred strains. Behavioural brain research 176 , 4-20. Nabavi, S., Fox, R., Proulx, C.D., Lin, J.Y., Tsien, R.Y., and Malinow, R. (2014). Engineering a memory with LTD and LTP. Nature 511 , 348-352. Nadler, J.V., Perry, B.W., and Cotman, C.W. (1978). Intraventricular kainic acid preferentially destroys hippocampal pyramidal cells. Nature 271 , 676-677. Nakashiba, T., Cushman, J.D., Pelkey, K.A., Renaudineau, S., Buhl, D.L., McHugh, T.J., Rodriguez Barrera, V., Chittajallu, R., Iwamoto, K.S., McBain, C.J. , et al. (2012). Young dentate 87 granule cells mediate pattern separation, whereas old granule cells facilitate pattern completion. Cell 149 , 188-201. Nakashiba, T., Young, J.Z., McHugh, T.J., Buhl, D.L., and Tonegawa, S. (2008). Transgenic inhibition of synaptic transmission reveals role of CA3 output in hippocampal learning. Science (New York, NY) 319 , 1260-1264. Naro, F., Donchenko, V., Minotti, S., Zolla, L., Molinaro, M., and Adamo, S. (1997). Role of phospholipase C and D signalling pathways in vasopressin-dependent myogenic differentiation. Journal of cellular physiology 171 , 34-42. Okuyama, T., Kitamura, T., Roy, D.S., Itohara, S., and Tonegawa, S. (2016). Ventral CA1 neurons store social memory. Science (New York, NY) 353 , 1536-1541. Oliveira, T.G., Chan, R.B., Tian, H., Laredo, M., Shui, G., Staniszewski, A., Zhang, H., Wang, L., Kim, T.W., Duff, K.E. , et al. (2010). Phospholipase d2 ablation ameliorates Alzheimer's diseaselinked synaptic dysfunction and cognitive deficits. The Journal of neuroscience : the official journal of the Society for Neuroscience 30 , 16419-16428. Pagani, J.H., Zhao, M., Cui, Z., Avram, S.K., Caruana, D.A., Dudek, S.M., and Young, W.S. (2015). Role of the vasopressin 1b receptor in rodent aggressive behavior and synaptic plasticity in hippocampal area CA2. Mol Psychiatry 20 , 490-499. Panda, A., Thakur, R., Krishnan, H., Naik, A., Shinde, D., and Raghu, P. (2018). Functional analysis of mammalian phospholipase D enzymes. Bioscience reports 38 . Pinto, V., Costa, J.C., Morgado, P., Mota, C., Miranda, A., Bravo, F.V., Oliveira, T.G., Cerqueira, J.J., and Sousa, N. (2015). Differential impact of chronic stress along the hippocampal dorsalventral axis. Brain structure & function 220 , 1205-1212. Piskorowski, R.A., Nasrallah, K., Diamantopoulou, A., Mukai, J., Hassan, S.I., Siegelbaum, S.A., Gogos, J.A., and Chevaleyre, V. (2016). Age-Dependent Specific Changes in Area CA2 of the Hippocampus and Social Memory Deficit in a Mouse Model of the 22q11.2 Deletion Syndrome. Neuron 89 , 163-176. Raam, T., McAvoy, K.M., Besnard, A., Veenema, A.H., and Sahay, A. (2017). Hippocampal oxytocin receptors are necessary for discrimination of social stimuli. Nature communications 8 , 2001. Roy, D.S., Kitamura, T., Okuyama, T., Ogawa, S.K., Sun, C., Obata, Y., Yoshiki, A., and Tonegawa, S. (2017). Distinct Neural Circuits for the Formation and Retrieval of Episodic Memories. Cell 170 , 1000-1012.e1019. Santa-Marinha, L., Castanho, I., Silva, R.R., Bravo, F.V., Miranda, A.M., Meira, T., Morais-Ribeiro, R., Marques, F., Xu, Y., Point du Jour, K. , et al. (2020). Phospholipase D1 Ablation Disrupts Mouse Longitudinal Hippocampal Axis Organization and Functioning. Cell reports 30 , 41974208.e4196. 88 Shah, S., Lubeck, E., Zhou, W., and Cai, L. (2017). seqFISH Accurately Detects Transcripts in Single Cells and Reveals Robust Spatial Organization in the Hippocampus. Neuron 94 , 752758.e751. Silva, B.A., Burns, A.M., and Gräff, J. (2019). A cFos activation map of remote fear memory attenuation. Psychopharmacology 236 , 369-381. Sloviter, R.S., and Damiano, B.P. (1981). Sustained electrical stimulation of the perforant path duplicates kainate-induced electrophysiological effects and hippocampal damage in rats. Neuroscience letters 24 , 279-284. Smith, A.S., Williams Avram, S.K., Cymerblit-Sabba, A., Song, J., and Young, W.S. (2016). Targeted activation of the hippocampal CA2 area strongly enhances social memory. Mol Psychiatry 21 , 1137-1144. Stackman, R.W., Jr., Cohen, S.J., Lora, J.C., and Rios, L.M. (2016). Temporary inactivation reveals that the CA1 region of the mouse dorsal hippocampus plays an equivalent role in the retrieval of long-term object memory and spatial memory. Neurobiology of learning and memory 133 , 118-128. Strange, B.A., Witter, M.P., Lein, E.S., and Moser, E.I. (2014). Functional organization of the hippocampal longitudinal axis. Nature reviews Neuroscience 15 , 655-669. Takayanagi, Y., Yoshida, M., Bielsky, I.F., Ross, H.E., Kawamata, M., Onaka, T., Yanagisawa, T., Kimura, T., Matzuk, M.M., Young, L.J. , et al. (2005). Pervasive social deficits, but normal parturition, in oxytocin receptor-deficient mice. Proceedings of the National Academy of Sciences of the United States of America 102 , 16096-16101. Talley, E.M., Solorzano, G., Lei, Q., Kim, D., and Bayliss, D.A. (2001). Cns distribution of members of the two-pore-domain (KCNK) potassium channel family. The Journal of neuroscience : the official journal of the Society for Neuroscience 21 , 7491-7505. Thompson, C.L., Pathak, S.D., Jeromin, A., Ng, L.L., MacPherson, C.R., Mortrud, M.T., Cusick, A., Riley, Z.L., Sunkin, S.M., Bernard, A. , et al. (2008). Genomic anatomy of the hippocampus. Neuron 60 , 1010-1021. Tsien, J.Z., Huerta, P.T., and Tonegawa, S. (1996). The essential role of hippocampal CA1 NMDA receptor-dependent synaptic plasticity in spatial memory. Cell 87 , 1327-1338. Vermeren, M.M., Zhang, Q., Smethurst, E., Segonds-Pichon, A., Schrewe, H., and Wakelam, M.J. (2016). The Phospholipase D2 Knock Out Mouse Has Ectopic Purkinje Cells and Suffers from Early Adult-Onset Anosmia. PloS one 11 , e0162814. Wang, W., Kiyoshi, C.M., Du, Y., Taylor, A.T., Sheehan, E.R., Wu, X., and Zhou, M. (2020). TREK1 Null Impairs Neuronal Excitability, Synaptic Plasticity, and Cognitive Function. Molecular neurobiology 57 , 1332-1346. Wersinger, S.R., Ginns, E.I., O'Carroll, A.M., Lolait, S.J., and Young, W.S., 3rd (2002). Vasopressin V1b receptor knockout reduces aggressive behavior in male mice. Mol Psychiatry 7 , 975-984. 89 Wersinger, S.R., Temple, J.L., Caldwell, H.K., and Young, W.S., 3rd (2008). Inactivation of the oxytocin and the vasopressin (Avp) 1b receptor genes, but not the Avp 1a receptor gene, differentially impairs the Bruce effect in laboratory mice (Mus musculus). Endocrinology 149 , 116-121. Yao, Y., Wang, X., Li, H., Fan, J., Qian, X., Li, H., and Xu, Y. (2020). Phospholipase D as a key modulator of cancer progression. Biological reviews of the Cambridge Philosophical Society. Yoshida, M., Takayanagi, Y., Inoue, K., Kimura, T., Young, L.J., Onaka, T., and Nishimori, K. (2009). Evidence that oxytocin exerts anxiolytic effects via oxytocin receptor expressed in serotonergic neurons in mice. The Journal of neuroscience : the official journal of the Society for Neuroscience 29 , 2259-2271. Young, W.S., Li, J., Wersinger, S.R., and Palkovits, M. (2006). The vasopressin 1b receptor is prominent in the hippocampal area CA2 where it is unaffected by restraint stress or adrenalectomy. Neuroscience 143 , 1031-1039. Zhang, W.N., Bast, T., and Feldon, J. (2001). The ventral hippocampus and fear conditioning in rats: different anterograde amnesias of fear after infusion of N-methyl-D-aspartate or its noncompetitive antagonist MK-801 into the ventral hippocampus. Behavioural brain research 126 , 159-174. Zhao, M., Choi, Y.S., Obrietan, K., and Dudek, S.M. (2007). Synaptic plasticity (and the lack thereof) in hippocampal CA2 neurons. The Journal of neuroscience : the official journal of the Society for Neuroscience 27 , 12025-12032. 96 residual PLD activity, better than the reconstitution with hPLD2 (Panda et al., 2018). Another interesting fact was that, when comparing the localization of dPLD, hPLD1, and hPLD2 in photoreceptors, dPLD and hPLD1 are uniquely distributed in the subplasma membrane region (Panda et al., 2018). PLD deficiency leads to apparent normal development of organisms (Bravo et al., 2018; Oliveira et al., 2010b; Panda et al., 2018). One possibility is that other sources of PA, such as diacylglycerol (DAG) kinases (DGK) and lyso-PA (LPA) acyltransferase (LPAAT), can compensate the lack of PLD (Oliveira and Di Paolo, 2010). But when we look closely to anatomical and physiological features, animal behavior and functioning under stress and disease, we observe relevant phenotypes upon PLD ablation. For example, yeast lacking the heterologous Pld gene do not exhibit any phenotype during vegetative growth, only during meiosis and sporulation (Rose et al., 1995). In the C. elegans , the lack of pld results in decreased levels of PA and PLD activity (Bravo et al., 2018) and PLD downregulation shortens longevity and induces age-related biomarkers through reactive oxygen species accumulation (Park et al., 2018). There are two PLD genes in zebrafish Danio rerio ( pld1 and pld2 ) (Raghu et al., 2009b) and manipulation of PLD1 in zebrafish results in intersegmental vascular pattern defects (Zeng et al., 2009). In Drosophila melanogaster the loss of function of the only Pld gene results in reduced PA levels (Panda et al., 2018) and in defects in vesicular transport that lead to collapse of the photoreceptor plasma membrane (Thakur et al., 2016). In mice, PLD ablation is responsible for behavior impairment (as discussed below) (Burkhardt et al., 2014). On the other hand, PLD overexpression also produces relevant phenotypes. It alters vesicular transport in cultured mammalian cells (Cai et al., 2006b; Choi et al., 2002; Huang et al., 2005; Vitale et al., 2001) and overexpression of dPLD in Drosophila results in a progressive collapse of the apical domain of these cells (retinal degeneration) (Panda et al., 2018). Also, overexpression of PLD1 decreases the levels of amyloid beta (Aβ) (Cai et al., 2006a) and promotes the formation of amyloid precursor protein (APP)- containing secretory vesicles from trans-Golgi network and inhibition of PLD1 activity decreases APP trafficking (Cai et al., 2006b), in the context of AD, as discussed below. In order to study the impact of PLD ablation, namely in the modulation of the longitudinal hippocampal axis, we made use of adult mice knock-out (KO) for Pld1 and/or Pld2 genes (we should have in mind that we cannot exclude the possibility that our results could be affected by an early developmental PLD deficits). We confirmed the lack of the corresponding isoenzyme in 97 these mice by western blot analysis, using an antibody that recognizes the COOH terminus of both PLD1 and PLD2 isoenzymes. Pld1 KO animals express only PLD2, Pld2 KO animals express only PLD1 enzyme, and Pld double-KO (DKO) animals have no PLD1 or PLD2 expression (see Chapter 2, figure 1A). As an approach to understand the differences between the two canonical mammalian PLD isoenzymes we used a method to study in vivo PLD activity performed by Oliveira et al (Oliveira et al., 2010b). By making use of the ability of PLD1 and PLD2 to use primary alcohols as nucleophiles over water in a transphosphatidylation, we injected mice with ethanol and then sacrificed to measure the levels of PEtOH with liquid chromatography-MS in the forebrain, in order to measure PLD activity (see Chapter 2, figures 1B and S1). With this experiment we confirmed that PLD1 and PLD2 are likely the only canonical PLD isoenzymes, since Pld DKO had almost no detectable PEtOH, indicating that PEtOH is exclusively produced by PLD1 and PLD2 and that the other PLD superfamily members (PLD3-6) do not contribute to the production of PEtOH (see Chapter 2, figures 1B and S1). Then, we conducted a broad-scale lipidomic profiling of DH and VH from both Pld1 and Pld2 WT and KO mice and analyzed the relative abundance of a total of 30 lipid classes, covering the 3 main lipid categories of sterols, sphingolipids, and glycerophospholipids (see Chapter 2, Figure 1C and Table S1). Once more we observed that PLD1 ablation creates a higher burden than PLD2 ablation, since PLD2 ablation did not have a significant impact on the total levels of the lipid classes analyzed (see Chapter 2, Figure 1C and Table S1) and PLD1 ablation significantly decreased the levels of several lipid classes (see Chapter 2, Figure 1C and Table S1), with a higher impact on the DH compared to VH, which is relatable with PLD1 protein levels being higher in the DH when compared to the VH (see Chapter 2, Figure S3B). The lack of PLD1 decreased the levels of PA, monoacylglycerol (MG) and lysoglycerophospholipids such as lysophosphatidylcholine (LPC), ether lysophosphatidylcholine (LPCe), and lysophosphatidylinositol (LPI) in the DH and the levels of PA and dihydroceramide (dhCer) in the VH (see Chapter 2, Figure 1C and Table S1). We should consider potential crosstalk between different phospholipases, that cleave the various bonds in phospholipids, namely phospholipase A (PLA2) superfamily that catalyze the hydrolysis of the sn-2 ester bond in a variety of different phospholipids, producing free fatty acids, such as arachidonic acid (AA), and lysophospholipids (Burke and Dennis, 2009), phospholipase C (PLC) that cleave the phosphodiester bond on the glycerol side forming diacylglycerols and a phospho-base, and PLD (Farooqui et al., 2000). Crosstalk between PLA2, PLC and PLD isozymes is crucial for maintaining normal homeostasis. 98 PLA2 reactions, resulting in free fatty acids, PLC reactions, resulting in DAG, and PLD catalyzed reactions, synergistically stimulate PKC activity (Farooqui and Horrocks, 2005). PLD and DAG generated by PLC stimulate various isoforms of protein kinase C (PKC), which activates both PLA2 and PLD (Clark et al., 1995; Farooqui and Horrocks, 2005). In addition, AA and eicosanoids produced by PLA2 activate isoforms of PLC, PLD, and PKC (Klein et al., 1995). In fact, coexpression of cytosolic (cPLA2) or type IIA secretory (sPLA2-IIA) PLA2 and PLD2, but not PLD1, augmented stimulus-induced arachidonate acid release (indicative of PLA2 activity) (Ueno et al., 2000). This justifies the observed lipidomic alterations in upon PLD1 ablation. While PLD2 ablation did not affect the levels of total PA in both DH and VH, PLD1 ablation led to a great decrease in the total levels of PA (see Chapter 2, Figure 1C and Table S1), in accordance with the observation of Pld1 KO animals having a greater decrease in PLD activity when compared to Pld2 KO animals (see Chapter 2, figure 1B) and in agreement with previous studies (Vermeren et al., 2016). The analysis of PA species upon ablation of either PLD1 or PLD2 revealed that Pld1 KO animals have decreased levels of nearly all PA species detected in both DH and VH, while PLD2 ablation leads to the decrease in only 3 PA species in the VH and an increase in 1 in DH, proving once more that PLD1 is a major contributor to total PA levels when compared to PLD2 (see Chapter 2, Figure 1D and Table S2). These findings indicate that PLD1 and PLD2 modulate different PA pools, which might account for or contribute to the differential functions of PLD1 and PLD2. Curiously, work from Panda et al. with overexpression of human PLD in a Drosophila model with dPLD loss of function ( dPLD3.1 ) also showed that hPLD1 and hPLD2 produce distinct molecular species of PA (Panda et al., 2018). They found that most of the PA species that were lower in dPLD3.1 were mostly reconstituted by dPLD (Panda et al., 2018). When reconstituted with human PLD, the pattern of species restored by PLD1 expression was substantially more similar to the observed in wild-type (WT) than that observed with overexpression of PLD2 (Panda et al., 2018). Different PA species exhibit different structure, which is related to the acyl chain length and unsaturation of the fatty acyl chain at sn-1 and sn-2 positions, leading to distinct repercussion on membrane structure, for example membrane curvature (van Meer, 2005). It was shown recently that PA saturation level influences its functions in neurosecretory pathway. While mono-unsaturated PA regulates the number of exocytotic events, poly-unsaturated PA regulates fusion pore stability and expansion (Tanguy et al., 2020a). Another interesting finding was that PLD1 affects both shorter and longer fatty acyl carbon length PA species, while PLD2 modulates predominantly shorter fatty acyl carbon length 99 PA species (see Chapter 2, Figures 1D and Table S2). It is known that shorter chain PA may promote membrane curvature and, curiously, PLD2 is primarily find in the plasma membrane. Several findings in our work also led us to hypothesize that there is a compensatory process by either PLD1 or PLD2 upon the other enzyme ablation: the sum of the decrease PEtOH production upon PLD1 and PLD2 ablation was less than 100% (see Chapter 2, figure 1B) and PLD2 ablation leads to increased levels of 4 PEtOH species (see Chapter 2, figure S1) and increased levels of one PA specie (see Chapter 2, figure 1D). We decided to explore the levels Pld1 /PLD1 and Pld2 /PLD2 mRNA (see Chapter 2, figure S2) and protein levels (see Chapter 2, figure S3) and conclude that ablating either of the PLD isoenzymes does not lead to expression upregulation of the other. One could consider other compensatory mechanisms such as localization modification, differences in co-factor modulation or post-translational modifications, such as histone acetylation, ubiquitination, phosphorylation or glycosylation. For example post-translational modification such as phosphorylation has been shown to regulate Spo14 localization and some bacterial toxins can regulate enzyme expression by modulating histone acetylation, which downregulates the NAPE-PLD (another human enzyme that exhibits PLD-like phosphodiesterase activity) promoter (Selvy et al., 2011). Moreover PLD activity in vivo is mediated by cellular calcium fluctuations and recruitment of PLD upon phosphatidylinositol bisphosphate or triphosphate (PIP2 or PIP3) production allows upstream lipid kinases or phosphatases to mediate PLD lipase activity (Selvy et al., 2011). Another fact is that PLD1 and PLD2 are phosphorylated in response to signal transduction as a regulatory mechanism and are post-translationally palmitoylated at two cysteine residues in the pleckstrin homology (PH) domain, which facilitates protein sorting into specific intracellular and plasma membrane domains including lipid rafts (Selvy et al., 2011). Interestingly, PLD1, but not PLD2, is post-translationally modified by multimonoubiquitination, which is important for modulating the localization and curbing lipase activity, and suppression of lipase activity either by mutation of the HKD motif or the phosphatidylinositol 4,5-bisphosphate binding motif or through use of PLD-selective inhibitors impairs the ubiquitination of PLD1 (Yin et al., 2010). Several methods have been used to study PLD activation and PLD-derived PA. Fluorescent protein fusions can localize PLD1 and PLD2, but do not differentiate their activation state. Genetically encoded probes directly visualize PA, although they cannot distinguish between different biosynthetic pools of PA. New tools for visualizing PLD signaling and PA are now emerging, in an attempt to tackle up and downstream PLD activation and the crosstalk and 100 compensation between PA synthesizing pathways. Raghu and colleagues applied a method of high performance liquid chromatography (HPLC)-coupled MS to determine the acyl chain composition of PA species at the sn −1 and sn −2 position, depending on ions generated by fragmentation of the corresponding PA anions that correspond to loss of one of the acyl chains. This enable assumption of the chain length and saturation of the individual fatty acid substituents in the parent phosphatidic acid molecule (Panda et al., 2018). It was also developed a chemoenzymatic, activity-based imaging method to visualize the precise subcellular locations of PLD activity with high spatiotemporal resolution, called IMPACT (Imaging Phospholipase D Activity with Clickable Alcohols via Transphosphatidylation) (Bumpus and Baskin, 2017). It relies on the PLD ability to accept azidoalcohols as reporters in a transphosphatidylation reaction and the resultant azidolipids are then fluorescently tagged, which allows visualization of cellular membranes bearing active PLD enzymes (Bumpus and Baskin, 2017). Moreover, the same laboratory created optogenetic controlled PLD (optoPLD), which is a complementary method for generating, rather than visualizing, organelle-specific PA pools (Tei and Baskin, 2020). Although PLD1 and PLD2 catalyze the same reaction, clearly the loss of function of one and/or another generate different repercussions on hippocampal lipidome and different phenotypes (see chapter 1), therefore dismantling the idea of absolute enzyme redundancy. PLD1 and/or PLD2 deficient mice have been proposed to show reduced brain growth at 14-27 days after birth (Burkhardt et al., 2014) and Pld2 KO mice have olfactory defects and an increased number of ectopic cerebellar Purkinje cells after 13 weeks of age (Vermeren et al., 2016). Moreover, Burkhardt et al. showed a deficit in the level of discrimination in long-term novel object recognition (NOR) test in Pld2 and double Pld KO and a social recognition deficit in Pld1 and/or Pld2 KO (Burkhardt et al., 2014). Other authors showed that PLD2 ablation had no major behavioral deficits: Oliveira et al., evaluated spatial working memory (using a radial arm water maze paradigm) and contextual fear conditioning memory (Oliveira et al., 2010b) and Vermeren at. al evaluated rotarod and open field on Pld2 KO animals (Vermeren et al., 2016). In chapter 2 (Santa-Marinha et al., 2020), we showed that PLD1 ablation impairs short-term object recognition memory and social exploration, since Pld1 KO mice were less social in a hippocampusdependent task, spending less time exploring compared to WT. In chapter 3, we showed that PLD2 ablation leads to social discrimination deficits with deficient ability to recognize a littermate and were less social in the same a hippocampus-dependent task. 101 Previously, Burkhardt et al. performed a partial characterization of behaviors upon ablation PLD1 and/or PLD2 in mice. Long term memory evaluated by NOR test was performed with a dayinterval between exposing the animal to two equal objects and then adding a third novel (Burkhardt et al., 2014) (different from our NOR test that tested short term memory with an intertrial of 1h). Short term memory evaluated as preference for social novelty was performed exposing an animal to an empty cage and a cage with an unknown mouse and then, after a 15 min break, a novel mouse was placed in previously empty cage and time interacting was counted (Burkhardt et al., 2014) (different from our preference for social novelty test with no inter-trial interval between the two trials). While Pld1 KO mice showed a trend toward a deficit in the level of discrimination in long-term NOR and a preference for social novelty deficit, PLD2 deficiency caused severe impairments on preference for social novelty and object recognition (Burkhardt et al., 2014). This work is not in total agreement with ours. Although PLD1 and PLD2 ablation led to decreased social exploration, the ability to prefer a novel mouse instead of a previously met (preference for social novelty) was not impaired. One potential caveat to consider as an explanation for the partial differences in observed results could be due to the different protocols used and to not using littermate mice as controls by Burkhardt et al.. Concerning the integration of the behavioral alterations with other phenotypic alterations observed by us, the social and object recognition deficits of Pld1 KO animals were associated with LTD decreased induction (See Chapter 2, Figure 4B, left panel) and decrease of synaptic proteins levels, namely SNAP-25 and GluN2A, in DH (see Chapter 2, Figure 5). Dorsal CA1 inhibition with muscimol, a selective GABAA receptor agonist that temporarily interrupt its function, was also shown to impair spatial and object memory (Stackman et al., 2016), although some studies conclude that the association of DH and object recognition is due to object-place recognition (Mendez et al., 2015; Oliveira et al., 2010a). It was also previously shown a role for CA1 and LTD in the acquisition of object–place configuration. Hippocampal LTD induction was associated with exploration of a new environment containing unfamiliar objects and/or familiar objects, whereas exploration of the new environment itself, in the absence of objects, impairs LTD (Kemp and Manahan-Vaughan, 2004). It agreement with our work, SNAP-25 heterozygous mice have a worse performance in the recognition of a novel object in a NOR protocol with 2h inter-trial interval and also have deficits in social behavior, with no deficits in spatial learning and memory tasks (Corradini et al., 2014). In addition to decreased levels of SNAP-25, Pld1 KO mice have reduced LTD induction in DH, which can also be caused by the reduced levels of SNAP-25. 102 In fact, infusion with a C-terminal SNAP-25 fragment (proposed to block its normal functions) reduces CA3-CA1 LTD induction, but not LTP (Zhang et al., 2011) and LTD is decreased in corticostriatal preparations of SNAP-25 heterozygous mice (Baca et al., 2013). Deficits in social behavior are also associated with impaired NMDAR-mediated neurotransmission, namely with GluN2A receptors (Zoicas and Kornhuber, 2019). For example, administratioin of PEAQX, a GluN2A-preferring antagonist, decreases social interaction in adolescent rats (Green et al., 2016; Morales and Spear, 2014). On the other hand, transgenic overexpression of GluN2A in forebrain, with an increased GluN2A: GluN2B ratio, leads to reduced long-term memory function and longterm memory impairments in a olfactory recognition task. (Jacobs and Tsien, 2014). The dynamic regulation and the combination of GluN2A and GluN2B subunits are crucial for physiological properties of NMDARs and should be evaluated. While the social and object recognition deficits caused by PLD1 ablation were correlated with the decrease of LTD (See Chapter 2, Figure 4B, left panel) and decrease of synaptic proteins in DH (see Chapter 2, Figure 5), the social deficits in Pld2 KO animals were associated with decreased LTP in VH (see Chapter 3, Figure 2A, right panel) without alterations in synaptic proteins (see Chapter 3, Figure 3). The lack of PLD2 did not interfere with sociability and preference for social novelty (see Chapter 3, Figure 1B, 1C, S1A and S1B) but Pld2 KO mice lost the ability to discriminate between a littermate and a strange mouse and spent less time interacting with conspecifics in this social discrimination task (see Chapter 3, Figure 1D and S1C). Curiously, the loss of the ability to discriminate between a littermate and a novel subject phenocopies the silencing of dorsal CA2 (Meira et al., 2018), proposing a mechanism of CA2 dysfunction in Pld2 KO animals. CA2 is a hippocampal region with distinct features from CA1 and CA3, from synaptic connectivity and electrophysiological characteristics to gene expression profile and relative resistance to cell death and resistance to damage from injury and synaptic plasticity (Dudek et al., 2016; Nadler et al., 1978; Sloviter and Damiano, 1981; Zhao et al., 2007). Several classes of proteins are particularly enriched in CA2. In fact, in situ hybridization revealed that PLD2 is highly expressed in CA2 when compared to CA1 and CA3 (Vermeren et al., 2016). Arginine vasopressin (AVP) and oxytocin (OXT) are two hypothalamic produced neuropeptides and AVP receptor 1b (AVPR1b) and OXT receptor (OXTR) also highly expressed in CA2 pyramidal neurons (Hammock and Levitt, 2013; Hidema et al., 2016; Mitre et al., 2016; Yoshida et al., 2009; Young et al., 2006). Modification or alteration on OXT/OXTR or AVPR1b have an impact on social behavior. Decreased levels of OXT and OXTR lead to social deficits and poor social 103 recognition (Lee et al., 2008; Takayanagi et al., 2005) and DG and CA2/CA3 OXTR are indispensable for discrimination of social stimuli (Raam et al., 2017) and long-term social recognition memory, without any effect on sociability and preference for social novelty (Lin et al., 2018). Moreover, AVPR1b KO mice display sociability and social memory impairments (DeVito et al., 2009; Wersinger et al., 2002; Wersinger et al., 2008). It is known that PLD activity is enhanced by AVP and OXT (Coletti et al., 2000; Inamori et al., 1995; Komati et al., 2005; Li et al., 2001; Morrison et al., 1996; Naro et al., 1997). In fact, AVP increases the PEtOH formation (indicating PLD activity) and inhibition of PLD decreases AVP produced PEtOH in cultures (Kusaka et al., 1996). Moreover, TWIK-related K+ channel (TREK) 1 is a PA-sensitive K2P channel that directly binds to PLD2 and, curiously, is also another protein highly expressed in CA2 (Talley et al., 2001). Importantly, PLD2 (and not PLD1) potentiates TREK1 current through local PA production that tonically activates the channel (Comoglio et al., 2014). An upregulated current of TREK1 channel in CA2 is found in Human 22q11.2 deletion syndrome (22q11.2DS) mouse model (Df(16)A+/−), also known as DiGeorge syndrome (the greatest molecular genetic risk factor for schizophrenia). These mice also show impaired social memory, phenocopying a silent CA2 (Piskorowski et al., 2016). As discussed below, TREK1 ablation in mice have decreased LTP in the CA1 region, similarly to VH of Pld2 KO mice, associated with recognition memory deficits (Wang et al., 2020). Considering these associations already described between PLD and AVP/OXT/TREK1, we can speculate a dysregulation on these pathways, possibly in CA2, were they are highly expressed, thus leading to the CA2 dependent impairment of littermate recall phenotype observed in Pld2 KO mice. One of the most important characteristics of the mammalian brain is its plasticity. It has been proposed that associative memories are stored as changes in the strength of synaptic connections between neurons, also called synaptic plasticity (Bannerman et al., 2014; Citri and Malenka, 2008). The intrinsic functionally of DH and VH can also be demonstrated by the different properties of synaptic plasticity. In our work, the DH and VH of WT mice presented different ability to produce a PP response (See Chapter 2, Figure 4C and Chapter 3, Figure 2C), a reliable signature already reported in rats (Maggio and Segal, 2007; Pinto et al., 2015). We can hypothesize that this intrinsic variability could be related with the intrinsic synaptic protein levels in DH and VH of WT animals (See Chapter 2, Figure 5). For example, whole-life exposure in rats to perfluorooctane sulfonate, which decreases the mRNA levels of AMPA receptor subunits GluA1 and GluA2, moderately suppresses PP facilitation (Zhang et al., 2019) and a study to aim the 104 adverse effects of copper oxide nanoparticles administration to rats revealed decrease of both PP and expression of GluN2A, but not GluN2B (Li et al., 2018) in CA1 region. We addressed the ex vivo impact of PLD removal in the trisynaptic circuit in the DH and VH poles. In chapter 2 we show that PLD1 ablation reduced LTD induction in the DH after lowfrequency stimulation (See Chapter 2, Figure 4B, left panel). Recently, through the use of inhibitors and Pld1 KO mice, it was shown that PLD1, but not PLD2, is necessary for M1 (subtype of muscarinic acetylcholine receptor)-LTD induction in the mouse prefrontal cortex (Moran et al., 2019), supporting our result of impairment of LTD in DH of Pld1 KO animals. Moreover, mechanistic target of rapamycin complex 1 (mTORC1) activation through PA derived from PLD signaling is a regulator of homeostatic signaling and PLD1 overexpression is sufficient to activate mTORC1 and alter synaptic function in cultured hippocampal neurons (Henry et al., 2018). Modulation of mTOR also modifies macroautophagy, altering the presynaptic structure and neurotransmission (Hernandez et al., 2012). The role of PLD1 and SNAP-25 has also been described in the autophagy field, since the induction of SNAP-25 in cancer cells maintains autophagic flux and autolysosomal efflux (Mu et al., 2018) and PLD1 was suggested to regulate autophagy through a possible effect in the VPS34/PI(3)P pathway (Dall'Armi et al., 2010). More recently, the PLD pathway was shown to modulate the autophagy in retinal pigment epithelium cells (Bermúdez et al., 2019). We can then speculate that the decreased levels of SNAP-25 in the DH of Pld1 KO animals may be an effector of the role of PLD1 as a modulator of autophagy. Electrophysiological changes in the DH of mice lacking PLD1 were accompanied by decreased levels of SNAP-25 and GluN2A. As previously discussed, the reduced LTD induction in DH of Pld1 KO mice could be caused by the decreased levels of SNAP-25, since blocking the normal functions of this protein reduces CA3-CA1 LTD induction (Zhang et al., 2011) and SNAP-25 heterozygous mice have decreased LTD in corticostriatal preparations (Baca et al., 2013). SNAP25 is subject to alternative splicing, producing SNAP-25a (predominant isoform in neurons in mice during embryonic and early postnatal development) and SNAP-25b (the dominant isoform as central synapses mature into adulthood) (Gopaul et al., 2020). In the first two weeks of development, rodents exhibit predominantly, or entirely, metabotropic glutamate receptor (mGluR)-dependent forms of LTD of synaptic transmission (Gopaul et al., 2020). With the change in relative levels of SNAP-25a to SNAP-25b there is upregulation NMDARs, suggesting that SNAP25a may play a greater role in the expression of mGluR-dependent LTD, while SNAP-25b may be more important for expression of NMDAR-dependent LTD and LTP (Gopaul et al., 2020). A recent 105 study showed that SNAP-25a favors the expression of LTD over LTP in the developing brain in one month old animals, while SNAP-25b deficient mice displayed similar LTD as their littermate controls (Gopaul et al., 2020). These results suggest that the balance between the two isoforms has an electrophysiological impact worthy of study in the future. Based on these recent observations, an important future experiment to be performed is to assess the balance of SNAP25a and b in Pld KO models, as wells as their interaction with PLD. The balance of the levels of GluN2A has also been subject of studies with alterations in hippocampal synaptic properties. For example, an increase in GluN2A levels selectively alters long term memory formation and, although it abolishes one type of protocol of LTD induction, it has no effects in a protocol similar to ours in CA1 from hippocampal slices (Cui et al., 2013). Another interesting fact is that it has been shown that LTD between the amygdala and perirhinal cortex is induced presynaptically via GluN2A-containing NMDARs (Laing and Bashir, 2015). NMDARs mediate LTP and LTD, but inconsistent results have been published, some of them highlighting the balance of GluN2A/GluN2B and others dismissing these results (Shipton and Paulsen, 2014; Wong and Gray, 2018). Once again, the balance of GluN2A/GluN2B is also an interesting line of research to be explored in Pld KO models. In chapter 3 we show that PLD2 ablation impairs LTP generation in the VH after tetanic stimulation (See Chapter 3, Figure 2A, right panel) at the Schaffer collaterals. Although previous work showed no differences in LTP upon PLD2 ablation in hippocampal slices, this experiment did not take into account DH-VH differentiation (Oliveira et al., 2010b). Moreover, dorsal CA2 (a region with high levels of PLD2, as discussed before) provides important social information to ventral CA1 to support social memory (Meira et al., 2018), a region that is impaired in Pld2 KO mice, with decreased LTP (see Chapter 3, Figure 2A, right panel). OXT and AVPR1b (highly expressed in CA2 as discussed before) are also implicated in synaptic plasticity. While OXT transiently reduces synaptic inhibition in multiple brain regions and enables long-term synaptic plasticity in the auditory cortex (Mitre et al., 2016), selective activation of AVPR1b or OXTR agonists induces significant potentiation in excitatory synaptic responses in CA2, despite its resistance to LTP (Pagani et al., 2015). Moreover, TREK1 total ablation in mice results in decreased LTP in CA1 region, which phenocopies PLD2 ablation (Wang et al., 2020). Taking into account the interaction of these proteins with PLD2, one should consider that these interactions can be affected in Pld2 KO mice, thus contributing to LTP impairment. 112 Box 2. Chapter 3 highlights. Future Perspectives Overall, we display the importance of PLD1/PLD2 as lipidic modulators of hippocampal longitudinal axis physiology, being essential regulators of hippocampal anatomy and synaptic functioning, which dysregulation leads to phenotypical impairment. We need to further explore compensatory mechanisms to the production of PA levels, either compensation of one isoenzyme by the other, by measuring in vivo PLD activity, either by other PA producing enzymes. Since in these studies we used genetic models to PLD1/2 levels, we plan to test in the future the phenotypical behavior and synaptic functions upon acute and chronic pharmacologic inhibition. It will be essential to explore the mechanism linking the lack of PLD2 (and also PLD1) with social behavior impairment. We look forward to tackle the relationship between OTR/AVP and their receptors, either by colocalization studies, protein quantification or acute inhibition of PLD2 and explore modifications in TREK1 induced currents. PLD1/PLD2 ablation is responsible for learning and memory defects, therefore it would be critical to explore the role of PLD in pathophysiology of diseases related to these deficits (namely AD and mood disorders, such as depression). 113 Since PLD is a risk factor and enhances the pathophysiology of several diseases, such as AD, and since PLD inhibition/ablation is compatible with life, it would be important to study PLD as a therapeutical target, in an attempt to ameliorate their pathological deficits. 114 References Ammar, M.R., Humeau, Y., Hanauer, A., Nieswandt, B., Bader, M.F., and Vitale, N. (2013). The Coffin-Lowry syndrome-associated protein RSK2 regulates neurite outgrowth through phosphorylation of phospholipase D1 (PLD1) and synthesis of phosphatidic acid. The Journal of neuroscience : the official journal of the Society for Neuroscience 33 , 19470-19479. Ammar, M.R., Thahouly, T., Hanauer, A., Stegner, D., Nieswandt, B., and Vitale, N. (2015). PLD1 participates in BDNF-induced signalling in cortical neurons. Scientific reports 5 , 14778. Anney, R., Klei, L., Pinto, D., Regan, R., Conroy, J., Magalhaes, T.R., Correia, C., Abrahams, B.S., Sykes, N., Pagnamenta, A.T. , et al. (2010). A genome-wide scan for common alleles affecting risk for autism. Human molecular genetics 19 , 4072-4082. Baca, M., Allan, A.M., Partridge, L.D., and Wilson, M.C. (2013). Gene-environment interactions affect long-term depression (LTD) through changes in dopamine receptor affinity in Snap25 deficient mice. Brain research 1532 , 85-98. Bannerman, D.M., Sprengel, R., Sanderson, D.J., McHugh, S.B., Rawlins, J.N., Monyer, H., and Seeburg, P.H. (2014). Hippocampal synaptic plasticity, spatial memory and anxiety. Nature reviews Neuroscience 15 , 181-192. Barber, C.N., Huganir, R.L., and Raben, D.M. (2018). Phosphatidic acid-producing enzymes regulating the synaptic vesicle cycle: Role for PLD? Adv Biol Regul 67 , 141-147. Bergin, D.H., Jing, Y., Mockett, B.G., Zhang, H., Abraham, W.C., and Liu, P. (2018). Altered plasma arginine metabolome precedes behavioural and brain arginine metabolomic profile changes in the APPswe/PS1ΔE9 mouse model of Alzheimer's disease. Transl Psychiatry 8 , 108. Bermúdez, V., Tenconi, P.E., Giusto, N.M., and Mateos, M.V. (2019). Lipopolysaccharide-Induced Autophagy Mediates Retinal Pigment Epithelium Cells Survival. Modulation by the Phospholipase D Pathway. Front Cell Neurosci 13 , 154. Biasibetti, R., Almeida Dos Santos, J.P., Rodrigues, L., Wartchow, K.M., Suardi, L.Z., Nardin, P., Selistre, N.G., Vázquez, D., and Gonçalves, C.A. (2017). Hippocampal changes in STZ-model of Alzheimer's disease are dependent on sex. Behavioural brain research 316 , 205-214. Bourne, K.Z., Natarajan, C., Perez, C.X.M., Tumurbaatar, B., Taglialatela, G., and Krishnan, B. (2019). Suppressing aberrant phospholipase D1 signaling in 3xTg Alzheimer's disease mouse model promotes synaptic resilience. Scientific reports 9 , 18342. Bradley, R.M., and Duncan, R.E. (2018). The lysophosphatidic acid acyltransferases (acylglycerophosphate acyltransferases) family: one reaction, five enzymes, many roles. Current opinion in lipidology 29 , 110-115. Bradley, R.M., Mardian, E.B., Bloemberg, D., Aristizabal Henao, J.J., Mitchell, A.S., Marvyn, P.M., Moes, K.A., Stark, K.D., Quadrilatero, J., and Duncan, R.E. (2017). Mice Deficient in lysophosphatidic acid acyltransferase delta (Lpaatδ)/acylglycerophosphate acyltransferase 4 (Agpat4) Have Impaired Learning and Memory. Molecular and cellular biology 37 . 115 Bravo, F.V., Da Silva, J., Chan, R.B., Di Paolo, G., Teixeira-Castro, A., and Oliveira, T.G. (2018). Phospholipase D functional ablation has a protective effect in an Alzheimer's disease Caenorhabditis elegans model. Scientific reports 8 , 3540. Bumpus, T.W., and Baskin, J.M. (2017). Clickable Substrate Mimics Enable Imaging of Phospholipase D Activity. ACS central science 3 , 1070-1077. Burke, J.E., and Dennis, E.A. (2009). Phospholipase A2 biochemistry. Cardiovascular drugs and therapy 23 , 49-59. Burkhardt, U., Stegner, D., Hattingen, E., Beyer, S., Nieswandt, B., and Klein, J. (2014). Impaired brain development and reduced cognitive function in phospholipase D-deficient mice. Neuroscience letters 572 , 48-52. Bustos, F.J., Varela-Nallar, L., Campos, M., Henriquez, B., Phillips, M., Opazo, C., Aguayo, L.G., Montecino, M., Constantine-Paton, M., Inestrosa, N.C. , et al. (2014). PSD95 suppresses dendritic arbor development in mature hippocampal neurons by occluding the clustering of NR2B-NMDA receptors. PloS one 9 , e94037. Cai, D., Netzer, W.J., Zhong, M., Lin, Y., Du, G., Frohman, M., Foster, D.A., Sisodia, S.S., Xu, H., Gorelick, F.S. , et al. (2006a). Presenilin-1 uses phospholipase D1 as a negative regulator of betaamyloid formation. Proceedings of the National Academy of Sciences of the United States of America 103 , 1941-1946. Cai, D., Zhong, M., Wang, R., Netzer, W.J., Shields, D., Zheng, H., Sisodia, S.S., Foster, D.A., Gorelick, F.S., Xu, H. , et al. (2006b). Phospholipase D1 corrects impaired betaAPP trafficking and neurite outgrowth in familial Alzheimer's disease-linked presenilin-1 mutant neurons. Proceedings of the National Academy of Sciences of the United States of America 103 , 19361940. Chan, R.B., Oliveira, T.G., Cortes, E.P., Honig, L.S., Duff, K.E., Small, S.A., Wenk, M.R., Shui, G., and Di Paolo, G. (2012). Comparative lipidomic analysis of mouse and human brain with Alzheimer disease. The Journal of biological chemistry 287 , 2678-2688. Chen, G., Chen, K.S., Knox, J., Inglis, J., Bernard, A., Martin, S.J., Justice, A., McConlogue, L., Games, D., Freedman, S.B. , et al. (2000). A learning deficit related to age and beta-amyloid plaques in a mouse model of Alzheimer's disease. Nature 408 , 975-979. Choi, W.S., Kim, Y.M., Combs, C., Frohman, M.A., and Beaven, M.A. (2002). Phospholipases D1 and D2 regulate different phases of exocytosis in mast cells. Journal of immunology (Baltimore, Md : 1950) 168 , 5682-5689. Citri, A., and Malenka, R.C. (2008). Synaptic plasticity: multiple forms, functions, and mechanisms. Neuropsychopharmacology : official publication of the American College of Neuropsychopharmacology 33 , 18-41. Clark, J.D., Schievella, A.R., Nalefski, E.A., and Lin, L.L. (1995). Cytosolic phospholipase A2. Journal of lipid mediators and cell signalling 12 , 83-117. 116 Coletti, D., Silvestroni, L., Naro, F., Molinaro, M., Adamo, S., and Palleschi, S. (2000). Vesiclemediated phosphatidylcholine reapposition to the plasma membrane following hormone-induced phospholipase D activation. Experimental cell research 256 , 94-104. Colley, W.C., Sung, T.C., Roll, R., Jenco, J., Hammond, S.M., Altshuller, Y., Bar-Sagi, D., Morris, A.J., and Frohman, M.A. (1997). Phospholipase D2, a distinct phospholipase D isoform with novel regulatory properties that provokes cytoskeletal reorganization. Current biology : CB 7 , 191201. Comoglio, Y., Levitz, J., Kienzler, M.A., Lesage, F., Isacoff, E.Y., and Sandoz, G. (2014). Phospholipase D2 specifically regulates TREK potassium channels via direct interaction and local production of phosphatidic acid. Proceedings of the National Academy of Sciences of the United States of America 111 , 13547-13552. Conde, M.A., Alza, N.P., Iglesias González, P.A., Scodelaro Bilbao, P.G., Sánchez Campos, S., Uranga, R.M., and Salvador, G.A. (2018). Phospholipase D1 downregulation by α-synuclein: Implications for neurodegeneration in Parkinson's disease. Biochimica et biophysica acta Molecular and cell biology of lipids 1863 , 639-650. Corradini, I., Donzelli, A., Antonucci, F., Welzl, H., Loos, M., Martucci, R., De Astis, S., Pattini, L., Inverardi, F., Wolfer, D. , et al. (2014). Epileptiform activity and cognitive deficits in SNAP-25(+/-) mice are normalized by antiepileptic drugs. Cerebral cortex (New York, NY : 1991) 24 , 364-376. Cruchaga, C., Karch, C.M., Jin, S.C., Benitez, B.A., Cai, Y., Guerreiro, R., Harari, O., Norton, J., Budde, J., Bertelsen, S. , et al. (2014). Rare coding variants in the phospholipase D3 gene confer risk for Alzheimer's disease. Nature 505 , 550-554. Cui, Z., Feng, R., Jacobs, S., Duan, Y., Wang, H., Cao, X., and Tsien, J.Z. (2013). Increased NR2A:NR2B ratio compresses long-term depression range and constrains long-term memory. Scientific reports 3 , 1036-1036. Dall'Armi, C., Hurtado-Lorenzo, A., Tian, H., Morel, E., Nezu, A., Chan, R.B., Yu, W.H., Robinson, K.S., Yeku, O., Small, S.A. , et al. (2010). The phospholipase D1 pathway modulates macroautophagy. Nature communications 1 , 142-142. DeVito, L.M., Konigsberg, R., Lykken, C., Sauvage, M., Young, W.S., 3rd, and Eichenbaum, H. (2009). Vasopressin 1b receptor knock-out impairs memory for temporal order. The Journal of neuroscience : the official journal of the Society for Neuroscience 29 , 2676-2683. Di Paolo, G., and Kim, T.W. (2011). Linking lipids to Alzheimer's disease: cholesterol and beyond. Nature reviews Neuroscience 12 , 284-296. Dudek, S.M., Alexander, G.M., and Farris, S. (2016). Rediscovering area CA2: unique properties and functions. Nature reviews Neuroscience 17 , 89-102. Farooqui, A.A., and Horrocks, L.A. (2005). Signaling and interplay mediated by phospholipases A2, C, and D in LA-N-1 cell nuclei. Reproduction, nutrition, development 45 , 613-631. 117 Farooqui, A.A., Horrocks, L.A., and Farooqui, T. (2000). Glycerophospholipids in brain: their metabolism, incorporation into membranes, functions, and involvement in neurological disorders. Chemistry and physics of lipids 106 , 1-29. Fazzari, P., Horre, K., Arranz, A.M., Frigerio, C.S., Saito, T., Saido, T.C., and De Strooper, B. (2017). PLD3 gene and processing of APP. Nature 541 , E1-e2. Frohman, M.A. (2015). The phospholipase D superfamily as therapeutic targets. Trends Pharmacol Sci 36 , 137-144. Gavin, A.L., Huang, D., Huber, C., Martensson, A., Tardif, V., Skog, P.D., Blane, T.R., Thinnes, T.C., Osborn, K., Chong, H.S. , et al. (2018). PLD3 and PLD4 are single-stranded acid exonucleases that regulate endosomal nucleic-acid sensing. Nature immunology 19 , 942-953. Glantz, L.A., and Lewis, D.A. (2000). Decreased dendritic spine density on prefrontal cortical pyramidal neurons in schizophrenia. Archives of general psychiatry 57 , 65-73. Gopaul, K.R., Irfan, M., Miry, O., Vose, L.R., Moghadam, A., Subah, G., Hökfelt, T., Bark, C., and Stanton, P.K. (2020). Developmental Time Course of SNAP-25 Isoforms Regulate Hippocampal Long-Term Synaptic Plasticity and Hippocampus-Dependent Learning. International journal of molecular sciences 21 . Gorbatyuk, O.S., Li, S., Nguyen, F.N., Manfredsson, F.P., Kondrikova, G., Sullivan, L.F., Meyers, C., Chen, W., Mandel, R.J., and Muzyczka, N. (2010). α-Synuclein expression in rat substantia nigra suppresses phospholipase D2 toxicity and nigral neurodegeneration. Molecular therapy : the journal of the American Society of Gene Therapy 18 , 1758-1768. Green, T.L., Burket, J.A., and Deutsch, S.I. (2016). Age-dependent effects on social interaction of NMDA GluN2A receptor subtype-selective antagonism. Brain research bulletin 125 , 159-167. Gustavsson, L. (1995). ESBRA 1994 Award Lecture. Phosphatidylethanol formation: specific effects of ethanol mediated via phospholipase D. Alcohol and alcoholism (Oxford, Oxfordshire) 30 , 391-406. Hammock, E.A., and Levitt, P. (2013). Oxytocin receptor ligand binding in embryonic tissue and postnatal brain development of the C57BL/6J mouse. Frontiers in behavioral neuroscience 7 , 195. Harris, J.A., Devidze, N., Halabisky, B., Lo, I., Thwin, M.T., Yu, G.Q., Bredesen, D.E., Masliah, E., and Mucke, L. (2010). Many neuronal and behavioral impairments in transgenic mouse models of Alzheimer's disease are independent of caspase cleavage of the amyloid precursor protein. The Journal of neuroscience : the official journal of the Society for Neuroscience 30 , 372-381. Henry, F.E., Wang, X., Serrano, D., Perez, A.S., Carruthers, C.J.L., Stuenkel, E.L., and Sutton, M.A. (2018). A Unique Homeostatic Signaling Pathway Links Synaptic Inactivity to Postsynaptic mTORC1. The Journal of neuroscience : the official journal of the Society for Neuroscience 38 , 2207-2225. 118 Hernandez, D., Torres, C.A., Setlik, W., Cebrián, C., Mosharov, E.V., Tang, G., Cheng, H.C., Kholodilov, N., Yarygina, O., Burke, R.E. , et al. (2012). Regulation of presynaptic neurotransmission by macroautophagy. Neuron 74 , 277-284. Hidema, S., Fukuda, T., Hiraoka, Y., Mizukami, H., Hayashi, R., Otsuka, A., Suzuki, S., Miyazaki, S., and Nishimori, K. (2016). Generation of Oxtr cDNA(HA)-Ires-Cre Mice for Gene Expression in an Oxytocin Receptor Specific Manner. Journal of cellular biochemistry 117 , 1099-1111. Hiroyama, M., and Exton, J.H. (2005). Localization and regulation of phospholipase D2 by ARF6. Journal of cellular biochemistry 95 , 149-164. Hsiao, Y.H., Chang, C.H., and Gean, P.W. (2018). Impact of social relationships on Alzheimer's memory impairment: mechanistic studies. Journal of biomedical science 25 , 3. Huang, P., Altshuller, Y.M., Hou, J.C., Pessin, J.E., and Frohman, M.A. (2005). Insulin-stimulated plasma membrane fusion of Glut4 glucose transporter-containing vesicles is regulated by phospholipase D1. Molecular biology of the cell 16 , 2614-2623. Hutsler, J.J., and Zhang, H. (2010). Increased dendritic spine densities on cortical projection neurons in autism spectrum disorders. Brain research 1309 , 83-94. Igarashi, M., Kozaki, S., Terakawa, S., Kawano, S., Ide, C., and Komiya, Y. (1996). Growth cone collapse and inhibition of neurite growth by Botulinum neurotoxin C1: a t-SNARE is involved in axonal growth. The Journal of cell biology 134 , 205-215. Inamori, K., Sagawa, N., Hasegawa, M., Itoh, H., Yano, J., and Mori, T. (1995). Activation of phospholipase D in cultured human amnion cells. Reproduction, fertility, and development 7 , 1591-1594. Jacobs, S.A., and Tsien, J.Z. (2014). Overexpression of the NR2A subunit in the forebrain impairs long-term social recognition and non-social olfactory memory. Genes, brain, and behavior 13 , 376-384. Jan, Y.N., and Jan, L.Y. (2010). Branching out: mechanisms of dendritic arborization. Nature reviews Neuroscience 11 , 316-328. Jenkins, G.M., and Frohman, M.A. (2005). Phospholipase D: a lipid centric review. Cellular and molecular life sciences : CMLS 62 , 2305-2316. Kanaho, Y., Funakoshi, Y., and Hasegawa, H. (2009). Phospholipase D signalling and its involvement in neurite outgrowth. Biochim Biophys Acta 1791 , 898-904. Kanfer, J.N., Singh, I.N., Pettegrew, J.W., McCartney, D.G., and Sorrentino, G. (1996). Phospholipid metabolism in Alzheimer's disease and in a human cholinergic cell. Journal of lipid mediators and cell signalling 14 , 361-363. Karch, C.M., and Goate, A.M. (2015). Alzheimer's disease risk genes and mechanisms of disease pathogenesis. Biological psychiatry 77 , 43-51. 119 Kemp, A., and Manahan-Vaughan, D. (2004). Hippocampal long-term depression and long-term potentiation encode different aspects of novelty acquisition. Proceedings of the National Academy of Sciences of the United States of America 101 , 8192-8197. Kennedy, D.P., and Adolphs, R. (2012). The social brain in psychiatric and neurological disorders. Trends in cognitive sciences 16 , 559-572. Klein, J., Chalifa, V., Liscovitch, M., and Löffelholz, K. (1995). Role of phospholipase D activation in nervous system physiology and pathophysiology. Journal of neurochemistry 65 , 1445-1455. Klevanski, M., Herrmann, U., Weyer, S.W., Fol, R., Cartier, N., Wolfer, D.P., Caldwell, J.H., Korte, M., and Müller, U.C. (2015). The APP Intracellular Domain Is Required for Normal Synaptic Morphology, Synaptic Plasticity, and Hippocampus-Dependent Behavior. The Journal of neuroscience : the official journal of the Society for Neuroscience 35 , 16018-16033. Komati, H., Naro, F., Mebarek, S., De Arcangelis, V., Adamo, S., Lagarde, M., Prigent, A.F., and Némoz, G. (2005). Phospholipase D is involved in myogenic differentiation through remodeling of actin cytoskeleton. Molecular biology of the cell 16 , 1232-1244. Kosel, F., Torres Munoz, P., Yang, J.R., Wong, A.A., and Franklin, T.B. (2019). Age-related changes in social behaviours in the 5xFAD mouse model of Alzheimer's disease. Behavioural brain research 362 , 160-172. Krishnan, B., Kayed, R., and Taglialatela, G. (2018). Elevated phospholipase D isoform 1 in Alzheimer's disease patients' hippocampus: Relevance to synaptic dysfunction and memory deficits. Alzheimer's & dementia (New York, N Y) 4 , 89-102. Kulkarni, V.A., and Firestein, B.L. (2012). The dendritic tree and brain disorders. Mol Cell Neurosci 50 , 10-20. Kusaka, I., Ishikawa, S.E., Higashiyama, M., Saito, T., Nagasaka, S., and Saito, T. (1996). The activation of phospholipase D participates in the mitogenic action of arginine vasopressin in cultured rat glomerular mesangial cells. Endocrinology 137 , 5421-5428. Laing, M.D., and Bashir, Z.I. (2015). Presynaptic NR2A-Containing NMDARs Are Required for LTD between the Amygdala and the Perirhinal Cortex: A Potential Mechanism for the Emotional Modulation of Memory? eNeuro 2 . Lalli, G., and Hall, A. (2005). Ral GTPases regulate neurite branching through GAP-43 and the exocyst complex. The Journal of cell biology 171 , 857-869. Lee, H.J., Caldwell, H.K., Macbeth, A.H., Tolu, S.G., and Young, W.S., 3rd (2008). A conditional knockout mouse line of the oxytocin receptor. Endocrinology 149 , 3256-3263. Li, W.Q., Luo, L.D., Hu, Z.W., Lyu, T.J., Cen, C., and Wang, Y. (2019). PLD1 promotes dendritic spine morphogenesis via activating PKD1. Mol Cell Neurosci 99 , 103394. Li, X., Sun, W., and An, L. (2018). Nano-CuO impairs spatial cognition associated with inhibiting hippocampal long-term potentiation via affecting glutamatergic neurotransmission in rats. Toxicology and industrial health 34 , 409-421. 120 Li, Y., Shiels, A.J., Maszak, G., and Byron, K.L. (2001). Vasopressin-stimulated Ca2+ spiking in vascular smooth muscle cells involves phospholipase D. American journal of physiology Heart and circulatory physiology 280 , H2658-2664. Lin, Y.T., Hsieh, T.Y., Tsai, T.C., Chen, C.C., Huang, C.C., and Hsu, K.S. (2018). Conditional Deletion of Hippocampal CA2/CA3a Oxytocin Receptors Impairs the Persistence of Long-Term Social Recognition Memory in Mice. The Journal of neuroscience : the official journal of the Society for Neuroscience 38 , 1218-1231. Lionel, A.C., Crosbie, J., Barbosa, N., Goodale, T., Thiruvahindrapuram, B., Rickaby, J., Gazzellone, M., Carson, A.R., Howe, J.L., Wang, Z. , et al. (2011). Rare copy number variation discovery and cross-disorder comparisons identify risk genes for ADHD. Science translational medicine 3 , 95ra75. Liscovitch, M., Czarny, M., Fiucci, G., Lavie, Y., and Tang, X. (1999). Localization and possible functions of phospholipase D isozymes. Biochim Biophys Acta 1439 , 245-263. Liu, Y., Zhang, Y.W., Wang, X., Zhang, H., You, X., Liao, F.F., and Xu, H. (2009). Intracellular trafficking of presenilin 1 is regulated by beta-amyloid precursor protein and phospholipase D1. The Journal of biological chemistry 284 , 12145-12152. Luo, L.D., Li, G., and Wang, Y. (2017). PLD1 promotes dendritic spine development by inhibiting ADAM10-mediated N-cadherin cleavage. Scientific reports 7 , 6035. Maggio, N., and Segal, M. (2007). Striking variations in corticosteroid modulation of long-term potentiation along the septotemporal axis of the hippocampus. The Journal of neuroscience : the official journal of the Society for Neuroscience 27 , 5757-5765. Meira, T., Leroy, F., Buss, E.W., Oliva, A., Park, J., and Siegelbaum, S.A. (2018). A hippocampal circuit linking dorsal CA2 to ventral CA1 critical for social memory dynamics. Nature communications 9 , 4163. Mendez, M., Arias, N., Uceda, S., and Arias, J.L. (2015). c-Fos expression correlates with performance on novel object and novel place recognition tests. Brain research bulletin 117 , 1623. Miranda, A.M., Bravo, F.V., Chan, R.B., Sousa, N., Di Paolo, G., and Oliveira, T.G. (2019). Differential lipid composition and regulation along the hippocampal longitudinal axis. Transl Psychiatry 9 , 144. Mitre, M., Marlin, B.J., Schiavo, J.K., Morina, E., Norden, S.E., Hackett, T.A., Aoki, C.J., Chao, M.V., and Froemke, R.C. (2016). A Distributed Network for Social Cognition Enriched for Oxytocin Receptors. The Journal of neuroscience : the official journal of the Society for Neuroscience 36 , 2517-2535. Morales, M., and Spear, L.P. (2014). The effects of an acute challenge with the NMDA receptor antagonists, MK-801, PEAQX, and ifenprodil, on social inhibition in adolescent and adult male rats. Psychopharmacology 231 , 1797-1807. 121 Moran, S.P., Xiang, Z., Doyle, C.A., Maksymetz, J., Lv, X., Faltin, S., Fisher, N.M., Niswender, C.M., Rook, J.M., Lindsley, C.W. , et al. (2019). Biased M(1) receptor-positive allosteric modulators reveal role of phospholipase D in M(1)-dependent rodent cortical plasticity. Science signaling 12 . Morrison, J.J., Dearn, S.R., Smith, S.K., and Ahmed, A. (1996). Activation of protein kinase C is required for oxytocin-induced contractility in human pregnant myometrium. Human reproduction (Oxford, England) 11 , 2285-2290. Mu, Y., Yan, X., Li, D., Zhao, D., Wang, L., Wang, X., Gao, D., Yang, J., Zhang, H., Li, Y. , et al. (2018). NUPR1 maintains autolysosomal efflux by activating SNAP25 transcription in cancer cells. Autophagy 14 , 654-670. Nadler, J.V., Perry, B.W., and Cotman, C.W. (1978). Intraventricular kainic acid preferentially destroys hippocampal pyramidal cells. Nature 271 , 676-677. Naro, F., Donchenko, V., Minotti, S., Zolla, L., Molinaro, M., and Adamo, S. (1997). Role of phospholipase C and D signalling pathways in vasopressin-dependent myogenic differentiation. Journal of cellular physiology 171 , 34-42. Nelson, R.K., and Frohman, M.A. (2015). Physiological and pathophysiological roles for phospholipase D. J Lipid Res 56 , 2229-2237. Nieweg, K., Schaller, H., and Pfrieger, F.W. (2009). Marked differences in cholesterol synthesis between neurons and glial cells from postnatal rats. Journal of neurochemistry 109 , 125-134. Oh, D.Y., Park, S.Y., Cho, J.H., Lee, K.S., Min do, S., and Han, J.S. (2007). Phospholipase D1 activation through Src and Ras is involved in basic fibroblast growth factor-induced neurite outgrowth of H19-7 cells. Journal of cellular biochemistry 101 , 221-234. Oliveira, A.M., Hawk, J.D., Abel, T., and Havekes, R. (2010a). Post-training reversible inactivation of the hippocampus enhances novel object recognition memory. Learning & memory (Cold Spring Harbor, NY) 17 , 155-160. Oliveira, T.G., Chan, R.B., Bravo, F.V., Miranda, A., Silva, R.R., Zhou, B., Marques, F., Pinto, V., Cerqueira, J.J., Di Paolo, G. , et al. (2016). The impact of chronic stress on the rat brain lipidome. Mol Psychiatry 21 , 80-88. Oliveira, T.G., Chan, R.B., Tian, H., Laredo, M., Shui, G., Staniszewski, A., Zhang, H., Wang, L., Kim, T.W., Duff, K.E. , et al. (2010b). Phospholipase d2 ablation ameliorates Alzheimer's diseaselinked synaptic dysfunction and cognitive deficits. The Journal of neuroscience : the official journal of the Society for Neuroscience 30 , 16419-16428. Oliveira, T.G., and Di Paolo, G. (2010). Phospholipase D in brain function and Alzheimer's disease. Biochim Biophys Acta 1801 , 799-805. Pagani, J.H., Zhao, M., Cui, Z., Avram, S.K., Caruana, D.A., Dudek, S.M., and Young, W.S. (2015). Role of the vasopressin 1b receptor in rodent aggressive behavior and synaptic plasticity in hippocampal area CA2. Mol Psychiatry 20 , 490-499.