Increased axonal bouton dynamics in the aging mouse cortex

Aging is a major risk factor for many neurological diseases and is associated with mild cognitive decline. Previous studies suggest that aging is accompanied by reduced synapse number and synaptic plasticity in specific brain regions. However, most studies, to date, used either postmortem or ex vivo preparations and lacked key in vivo evidence. Thus, whether neuronal arbors and synaptic structures remain dynamic in the intact aged brain and whether specific synaptic deficits arise during aging remains unknown. Here we used in vivo two-photon imaging and a unique analysis method to rigorously measure and track the size and location of axonal boutons in aged mice. Unexpectedly, the aged cortex shows circuit-specific increased rates of axonal bouton formation, elimination, and destabilization. Compared with the young adult brain, large (i.e., strong) boutons show 10-fold higher rates of destabilization and 20-fold higher turnover in the aged cortex. Size fluctuations of persistent boutons, believed to encode long-term memories, also are larger in the aged brain, whereas bouton size and density are not affected. Our data uncover a striking and unexpected increase in axonal bouton dynamics in the aged cortex. The increased turnover and destabilization rates of large boutons indicate that learning and memory deficits in the aged brain arise not through an inability to form new synapses but rather through decreased synaptic tenacity. Overall our study suggests that increased synaptic structural dynamics in specific cortical circuits may be a mechanism for age-related cognitive decline

Sequential neuromodulation of Hebbian plasticity offers mechanism for effective reward-based navigation

Spike timing-dependent plasticity (STDP) is under neuromodulatory control, which is correlated with distinct behavioral states. Previously we reported that dopamine, a reward signal, broadens the time window for synaptic potentiation and modulates the outcome of hippocampal STDP even when applied after the plasticity induction protocol (Brzosko et al., 2015). Here we demonstrate that sequential neuromodulation of STDP by acetylcholine and dopamine offers an efficacious model of reward-based navigation. Specifically, our experimental data in mouse hippocampal slices show that acetylcholine biases STDP towards synaptic depression, whilst subsequent application of dopamine converts this depression into potentiation. Incorporating this bidirectional neuromodulation-enabled correlational synaptic learning rule into a computational model yields effective navigation towards changing reward locations, as in natural foraging behavior. Thus, temporally sequenced neuromodulation of STDP enables associations to be made between actions and outcomes and also provides a possible mechanism for aligning the time scales of cellular and behavioral learning.Medical Research Council Studentship Zuzanna Brzosko Wolfram Schultz Ole Paulsen Engineering and Physical Sciences Research Council Studentship Sara Zannone Claudia Clopath Wellcome 095495 Wolfram Schultz Biotechnology and Biological Sciences Research Council BB/N013956/1 Claudia Clopath Wellcome 200790/Z/16/Z Claudia Clopath Biotechnology and Biological Sciences Research Council BB/N019008/1 Ole Paulse

Characterisation of new factors involved in invasion and survival of Toxoplasma gondii

Mon projet principal de thèse est consacré à la compréhension du rôle de ROP55, une nouvelle protéine de T.gondii, le parasite responsable de la toxoplasmose. ROP55 a été identifiée lors d'un criblage in-silico. Elle est présente dans le bulbe des rhoptries, des organelles de sécrétion nécessaires à l’invasion et la virulence des parasites. Nous avons découvert que l'infection de fibroblastes humains par des parasites mutants KOROP55, entraînait une augmentation de la mortalité des cellules hôtes, et une diminution de la parasitémie. L’analyse transcriptomique des fibroblastes humains infectés par les tachyzoïtes KOROP55 révèle un enrichissement des gènes caractéristiques d'une réponse pro-inflammatoire. De plus, nous avons démontré que les parasites KO ROP55 induisaient l'activation de NF-kB- un régulateur de la survie cellulaire et de la réponse inflammatoire - qui s'accompagnait de la sécrétion de la cytokine pro-inflammatoire IL-1B. L'effet de ROP55 sur la sécrétion d'IL-1B et la mort cellulaire est également conservé dans des cellules immunitaires humaines et de souris. La mort de la cellule hôte couplée à la sécrétion d'IL-1B est caractéristique d'un type de mort cellulaire régulée, appelée pyroptose. Au cours de ce type de mort cellulaire, la maturation et la sécrétion de l'IL-1B nécessitent l'activité protéolytique des caspases inflammatoires 1, 4 ou 5. Nous avons découvert que la caspase 1 était nécessaire à la sécrétion de l'IL-1B à partir de monocytes humains infectés par KOROP55, et que la caspase 8 était également impliquée. La caspase-8 est généralement considérée comme une caspase apoptotique, ce qui suggère que le phénotype de mort cellulaire observé lors de l'infection par des parasites de KOROP55 n'est pas la mort pyroptotique classique. Le mécanisme précis de la mort induite par les parasites KO ROP55 reste à caractériser.Enfin, en utilisant un modèle expérimental murin, nous avons montré que ROP55 est un important facteur de virulence in vivo. Alors qu’il suffit d'infecter des souris avec un seul parasite de type I pour induire la mortalité, l'infection de souris par 1 million de tachyzoïtes KO ROP55 n’entraîne la mort que de 50 % des souris. En outre, l'infection de souris avec une dose sub-létale de tachyzoïtes KOROP55 induit une protection contre une infection ultérieure par des parasites virulents.En conclusion, nous avons identifié ROP55 comme un nouvel effecteur parasitaire et facteur de virulence, qui supprime la réponse inflammatoire de l'hôte et maintient la survie des cellules de l'hôte, permettant une réplication et une propagation efficaces du parasite.En parallèle de l’étude de ROP55, j’ai initié la caractérisation du processus d'invasion des bradyzoïtes. En effet, l'invasion de T.gondii a été largement étudiée et caractérisée au stade tachyzoïte et de manière plus sommaire chez les sporozoïtes, mais le processus n'a jamais été investigué chez les bradyzoïtes. L’invasion des tachyzoïtes implique la formation d'une jonction mobile (MJ), composée des protéines AMA1, RON2, RON4, RON4L1, RON5 et RON8. Ces protéines sont sécrétées à partir des micronèmes (AMA1) et des rhoptries (RONs). AMA1 et RON2 interagissent étroitement pour permettent l’assemblage de la JM. Deux autres complexes, AMA3-RON2L2 et AMA4-RON2L1 sont présents chez les sporozoïtes. Les données de transcriptomique montrent qu’à l’exception de AMA3 et RON2L2, les gènes codant pour ces protéines sont fortement exprimés chez les bradyzoïtes. Nous avons confirmé la présence de toutes ces protéines dans les bradyzoïtes enkystés, et pu distinguer le marquage caractéristique en forme d'anneau de la MJ avec des anticorps dirigés contre RON2, RON4, RON5 et RON8. Ceci représente la première démonstration que les bradyzoïtes forment une MJ pour l'invasion des cellules hôtes. Il est intéressant de noter que nous avons pu observer également AMA4 à la MJ, suggérant que le complexe AMA4-RON2L1 participe également à l’invasion des bradyzoïtes.My thesis consists of two independent projects, which have been presented and discussed separately.My main project is dedicated to understanding the role of a new rhoptry bulb protein, ROP55, identified during an in-silico screen performed in the lab. We found that ROP55 is a Toxoplasma virulence factor, and that infecting human fibroblasts with KO ROP55 parasites, led to increased host cell mortality, and a decrease in parasitaemia. In order to gain an insight into this phenotype, we carried out RNA sequencing analysis of human fibroblasts infected with KO ROP55 tachyzoites and observed an enrichment of genes characteristic of a pro-inflammatory response. Furthermore, we demonstrated that KO ROP55 parasites induce the activation of NF-kB, a master regulator of cell survival and the inflammatory response, which is accompanied by the secretion of the proinflammatory cytokine IL-1B;. The effect of ROP55 on IL-1B; secretion and cell death was conserved in both human and mouse immune cells.Host cell death coupled with the secretion of IL-1B; is characteristic of a type of regulated cell death called pyroptosis. During pyroptotic cell death, the maturation and secretion of IL-1B; requires the proteolytic activity of the inflammatory caspases 1, 4 or 5. We found that caspase 1 and 8 were necessary for IL-1B; secretion from human monocytes infected with KO ROP55. Caspase-8 is typically considered an apoptotic caspase, suggesting that the cell death phenotype observed upon infection with KO ROP55 parasites is not the classical pyroptotic death. The precise death mechanism induced by KO ROP55 parasites remains to be characterized.Finally, we confirmed that ROP55 is an important virulence factor in-vivo using a mouse model. Infecting mice with a single wild-type, type I parasite, is sufficient to induce mortality. In contrast, infecting mice with 1 million KO ROP55 tachyzoites led to the death of only 50% of mice. Furthermore, infecting mice with a sub-lethal dose of KO ROP55 tachyzoites induced protection from a subsequent infection with virulent parasites. In conclusion, we identified ROP55 as a new parasite effector and virulence factor, which suppresses the host inflammatory response and maintains host cell survival, allowing efficient parasite replication and propagation.My second project focussed on the characterization of the process of invasion in bradyzoites. Although invasion has been extensively studied and characterized in the tachyzoite stage, the process has never been characterized in bradyzoites. Invasion by tachyzoites involves the assembly of a moving junction (MJ), composed of AMA1, RON2, RON4, RON4L1, RON5 and RON8. Transcriptomics data show that the genes encoding these proteins are highly expressed in bradyzoites, alongside AMA4 and RON2L1 which form a complex during invasion by sporozoites. We confirmed the presence of these proteins in encysted bradyzoites, and intriguingly, we could distinguish the characteristic ring shape labelling of the MJ with antibodies directed against RON2, RON4, RON5 and RON8. This represents the first demonstration that bradyzoites require a MJ during invasion of host cells. Interestingly, we could observe AMA4 at the MJ, suggesting that the AMA4-RON2L1 complex could be important for successful bradyzoite invasion

Cholinergic modulation of hippocampal network function

Cholinergic septohippocampal projections from the medial septal area to the hippocampus are proposed to have important roles in cognition by modulating properties of the hippocampal network. However, the precise spatial and temporal profile of acetylcholine release in the hippocampus remains unclear making it difficult to define specific roles for cholinergic transmission in hippocampal dependent behaviors. This is partly due to a lack of tools enabling specific intervention in, and recording of, cholinergic transmission. Here, we review the organization of septohippocampal cholinergic projections and hippocampal acetylcholine receptors as well as the role of cholinergic transmission in modulating cellular excitability, synaptic plasticity, and rhythmic network oscillations. We point to a number of open questions that remain unanswered and discuss the potential for recently developed techniques to provide a radical reappraisal of the function of cholinergic inputs to the hippocampus

Long-Term Depression of Synaptic Kainate Receptors Reduces Excitability by Relieving Inhibition of the Slow Afterhyperpolarization

Kainate receptors (KARs) are ionotropic glutamate receptors that also activate non-canonical G-protein-coupled signalling pathways to depress the slow afterhyperpolarization (sAHP). Here we show that long-term depression of KAR-mediated synaptic transmission (KAR LTD) at rat hippocampal mossy fiber synapses relieves inhibition of the sAHP by synaptic transmission. KAR LTD is induced by high frequency mossy fiber stimulation and natural spike patterns and requires activation of adenosine A(2A) receptors. Natural spike patterns also cause long-term potentiation of NMDA receptor-mediated synaptic transmission that overrides the effects of KAR LTD during low frequency synaptic input. However, KAR LTD is dominant at higher frequency synaptic stimulation where it decreases the cellular response by relieving inhibition of the sAHP. Thus we describe a form of glutamate receptor plasticity induced by natural spike patterns whose primary physiological function is to regulate cellular excitability

Spine calcium model

MATLAB code for spine calcium mode

PURE-LET denoising code

MATLAB code for PURE-LET denoising of spine calcium transient