Análisis de una topología bioinspirada en la Oliva Inferior

Máster Universitario en Investigación e Innovación en TIC (I2-TIC)Las neuronas de la Oliva Inferior (Inferior Olive, IO, en inglés) se caracterizan por tener actividad en forma de spike y oscilaciones subumbrales. Estas neuronas están acopladas mediante conexiones eléctricas (gap junctions) lo cual permite la sincronización entre ellas. En función de este acoplamiento, se generan ciertos patrones en la red que determinan en comportamiento de ésta y la generación de patrones espacio-temporales. En este trabajo de fin de máster se han simulado modelos de la IO con diferentes topologías de red teóricas que ya habían sido estudiadas con anterioridad, realizando una comparación entre la dinámica global de la red en cada una de ellas en función de distintos parámetros (grado de acoplamiento y número de conexiones entre neuronas). Además, se ha propuesto una nueva topología biológicamente inspirada basada en trabajos experimentales, comparando los resultados obtenidos en redes con esta topología con los observados en las topologías teóricas. Los resultados obtenidos nos muestran que hay tres parámetros fundamentales en la IO para la propagación del estímulo eléctrico, que son la fuerza de acoplamiento, el número de conexiones establecidas y la topología de red. Aplicando distintos valores a estos parámetros se observa que el comportamiento de la red de la IO es distinto. Una fuerza de acoplamiento mayor, así como un mayor número de conexiones implica una mayor sincronización de las neuronas de la red tanto en las oscilaciones subumbrales como en la actividad spiking, mientras que una fuerza de acoplamiento débil hace que cada neurona se comporte de una manera casi independiente sin conseguir esta sincronización. Por su parte, aplicando distintas topologías y manteniendo los otros dos parámetros también se ven diferencias de comportamiento, con la misma fuerza de acoplamiento en una topología bioinspirada, la sincronización que se genera es mayor que en una teórica y, además, la complejidad de los patrones generados aumenta creando distintos frentes de onda que compiten en sentidos opuestos

Generation of the complex spike in cerebellar Purkinje cells.

Each neuron of the nervous system is a machine specialised to appropriately transform its synaptic inputs into a pattern of spiking output. This is achieved through the combination of specialisations in synaptic properties and location, passive cell geometry and placement of particular active ion channels. The challenge presented to the neuroscientist is to, within each cell type, identify such specialisations in input distribution and resulting active events, and assess their relative importance in the generation of action potential output patterns. The Purkinje cell, in particular its response to climbing fibre (CF) input, is an excellent setting in which to attempt to meet this challenge. The Purkinje cell receives a single, easily isolated CF axon, which makes hundreds of synapses across the cell's highly branched, active dendritic tree, resulting in the generation of prominent dendritic calcium spikes and a distinctive, reproducible burst of fast action potentials (the complex spike) at the soma. In this thesis I have separated out the importance of the size of this input, its location and the active dendritic spikes it triggers in the generation of the complex spike. I have found that, to a large extent, the complex spike pattern is determined by the size of the CF input alone. I have characterised the complex spike (its number of spikes, their timing, height and reliability) at both constant physiological frequency and across a range of paired- pulse depression causing intervals. By alternating between whole cell current and voltage clamp in the same cell, I have recorded both the complex spikes and EPSCs generated at certain paired pulse intervals. In this way I have been able to construct the EPSC - complex spike 'input - output' relationship. This demonstrated that there is a straightforward linear transformation between the EPSC input amplitude and the number and timing of spikes in the complex spike. This applies across cells, explaining a large amount of the inter-cell variability in complex spike pattern. Input location and dendritic spikes have surprisingly little influence over the Purkinje cell complex spike. I found that complex spikes generated by dendritically distributed CF input can be reproduced by using conductance clamp to inject CF-like synaptic conductance at the soma. Both CF input and somatic EPSG injection produced complex spike waveforms that can only be easily explained by a model in which spikelets are initiated at a distant site and variably propagated to the soma. By using simultaneous somatic and dendritic recording I have demonstrated that this distant site initiation site is not in the dendrites. Somatic EPSG injection reproduced complex spikes independently of dendritic spikes, and extra dendritic spikes triggered by CF stimulation were associated with only 0.24 0.09 extra somatic spikelets in the complex spike. Rather, I have found that dendritic spikes, generated reliably by the dendritic location of CF inputs, have a role in regulating the post-complex spike pause. An extra dendritic spike generates a 3.4 0.7 mV deeper AHP and a 52 11 % longer pause before spontaneous spiking resumed. In this way, I have identified specialisations that encode the size, and thus timing, of CF inputs in the complex spike burst, whilst allowing the dendritic excitation of Purkinje cells (which is strongly associated synaptic and intrinsic plasticity) to be simultaneously encoded in the post-complex spike pause. This may reflect the complex spike's proposed dual role in both controlling ongoing movement and correcting for motor errors

29th Annual Computational Neuroscience Meeting: CNS*2020

Meeting abstracts This publication was funded by OCNS. The Supplement Editors declare that they have no competing interests. Virtual | 18-22 July 202

Intracellular processing of motion information in a network of blowfly visual interneurons

In the past few decades, the lobula plate of the fly has emerged as one of the leading models for the neural processing of optic flow stimuli that give rise to visual orientation behaviors (for recent reviews see Borst and Haag, 2002; Egelhaaf et al., 2002; Egelhaaf et al., 2002; Borst and Haag, 2007). The relative simplicity and accessibility of this neural system allows researchers to characterize the neural mechanisms that are thought to link the visual stimuli and the resulting behavioral responses. In the lobula plate, a set of 60 motion sensitive lobula plate tangential cells (LPTCs) integrate visual motion information from an array of local motion detectors, which form a retinotopic map of the fly’s visual space in the lobula plate. The selective pooling of local, direction selective inputs, together with a network of unilateral and bilateral interactions between LPTCs, shape and tune the response properties of LPTCs to behaviorally relevant optic flow stimuli. Over the years, lobula plate researchers assembled a formidable array of measurement and perturbation techniques that are usually available only in in-vitro systems. Additionally, the lobula plate and its presynaptic circuitry have been the subject of extensive and detailed modeling which allows a deeper synthetic understanding of the empirical results, as well as a more efficient and detailed way to generate hypotheses. In this work I used a selection of these tools to explore the role of intracellular processing of visual motion information in lobula plate neurons and the significance of spatial segregation and aggregation of these cells’ inputs in the context of their sensory function. Previous work on a network of ten LPTCs of the vertical system (VS cells) resulted in a prediction that due to lateral, gap-junction coupling of neighboring VS cells in their axon-terminals, the receptive fields of these cells should be broader in the axonal region than in the dendritic regions. I tested and confirmed this prediction using in-vivo calcium imaging and intracellular recordings. Using single-electrode voltage clamp I was able to perturb the flow of information in these cells and isolate the source of input responsible for this broadening, confirming that the coupling indeed takes place in the axon terminal. The separation of feed-forward, synaptic input in the dendrites from lateral, gap-junction coupling in the axon-terminals allowed me to experimentally ask what is the function of the receptive field broadening. Relying on model predictions, I showed that this broadening results in a more stable and smooth representation of optic flow in the output region of the cells than in their input region, when the fly is presented with naturalistic, patchy and non-uniform stimuli. I then showed, using a simplified compartmental model that the separation of axonal gap-junctions from the dendritic synaptic input makes the gap-junction coupling more effective, and is thus necessary to ensure the functionality of the lateral interactions

Activation of the pro-resolving receptor Fpr2 attenuates inflammatory microglial activation

Poster number: P-T099 Theme: Neurodegenerative disorders & ageing Activation of the pro-resolving receptor Fpr2 reverses inflammatory microglial activation Authors: Edward S Wickstead - Life Science & Technology University of Westminster/Queen Mary University of London Inflammation is a major contributor to many neurodegenerative disease (Heneka et al. 2015). Microglia, as the resident immune cells of the brain and spinal cord, provide the first line of immunological defence, but can become deleterious when chronically activated, triggering extensive neuronal damage (Cunningham, 2013). Dampening or even reversing this activation may provide neuronal protection against chronic inflammatory damage. The aim of this study was to determine whether lipopolysaccharide (LPS)-induced inflammation could be abrogated through activation of the receptor Fpr2, known to play an important role in peripheral inflammatory resolution. Immortalised murine microglia (BV2 cell line) were stimulated with LPS (50ng/ml) for 1 hour prior to the treatment with one of two Fpr2 ligands, either Cpd43 or Quin-C1 (both 100nM), and production of nitric oxide (NO), tumour necrosis factor alpha (TNFα) and interleukin-10 (IL-10) were monitored after 24h and 48h. Treatment with either Fpr2 ligand significantly suppressed LPS-induced production of NO or TNFα after both 24h and 48h exposure, moreover Fpr2 ligand treatment significantly enhanced production of IL-10 48h post-LPS treatment. As we have previously shown Fpr2 to be coupled to a number of intracellular signaling pathways (Cooray et al. 2013), we investigated potential signaling responses. Western blot analysis revealed no activation of ERK1/2, but identified a rapid and potent activation of p38 MAP kinase in BV2 microglia following stimulation with Fpr2 ligands. Together, these data indicate the possibility of exploiting immunomodulatory strategies for the treatment of neurological diseases, and highlight in particular the important potential of resolution mechanisms as novel therapeutic targets in neuroinflammation. References Cooray SN et al. (2013). Proc Natl Acad Sci U S A 110: 18232-7. Cunningham C (2013). Glia 61: 71-90. Heneka MT et al. (2015). Lancet Neurol 14: 388-40

Dynamics of evoked and spontaneous calcium transients in synaptic boutons of neocortical pyramidal neurons

In response to an action potential (AP), a transient rise in the intracellular calcium concentration ([Ca2+]i) causes transmitter release from nerve terminals. As the spatiotemporal dynamics of this calcium rise can affect the efficacy and plasticity of synaptic connections, it is essential to understand their determinants. To characterise factors that shape calcium transients in neocortical synaptic boutons, layer 5 pyramidal cells in the rat somatosensory cortex were filled through the patch pipette with a fluorescent calcium indicator for the measurement of [Ca2+]i. For accurate calculation of [Ca2+]i from the fluorescence intensity, the calcium binding affinities (Kd) of the indicators were measured in vitro, in solutions that were similar to the patch-clamp internal solution. These solutions were made with various concentrations of calcium chloride, but a constant concentration of a calcium buffer. The resultant free [Ca2+] was measured with a calcium-selective macroelectrode. It was found that the Kd values of the calcium indicators were considerably different from those previously published or provided by the manufacturers. Two main determinants of the intracellular calcium dynamics are the capacity of endogenous calcium buffers and the activity of calcium sequestration mechanisms. By measuring the peak amplitude of single AP-evoked calcium transients with different concentrations of OGB-1 or OGB-6F, a value of 7 was estimated for the calcium-binding ratio of endogenous buffers. Thus, in response to a single AP and in the absence of exogenous buffers, [Ca2+]i was raised by 5.3 microM, with a total change of approximately 50 microM. The rate constant of calcium sequestration (0.60 per s) was estimated from the slow decay time constant of the measured transients. The initial fast decay did not prolong when intracellular calcium uptake was inhibited, or speed up during repetitive stimulation. These findings suggest that calcium-induced calcium release (CICR), buffer saturation, and a non-linear calcium transporter were not the main cause of the bi-exponential decay. A 3D model of a bouton en passant showed that diffusion of calcium into the axon was likely the underlying mechanism. During high-frequency stimulation, CICR contributed to a supralinear summation of [Ca2+]i. Spontaneous increases in [Ca2+]i have been observed in several nerve terminals. They have been implicated in a number of cellular processes, including calcium homeostasis and spontaneous transmitter release. Here, the high-affinity calcium indicator OGB-1 was used to monitor small changes in [Ca2+]i. Spontaneous calcium transients (sCaTs) were observed at a frequency of around 0.2 per min. The increase in [Ca2+]i associated with each sCaT was 1.4–2.3 microM, in the absence of exogenous buffers. It was hypothesised that sCaTs arose from calcium release from presynaptic stores. In support of this, caffeine increased the average frequency of sCaTs by approximately 90%. The amplitude and kinetics of sCaTs identified in caffeine and in the control condition were not different from each other, suggesting that the majority of sCaTs might have been a result of calcium release through ryanodine receptors. The functional consequence(s) of sCaTs in neocortical synaptic boutons remains to be determined