25 research outputs found

    Pattern recognition of neural data: methods and algorithms for spike sorting and their optimal performance in prefrontal cortex recordings

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    Programa de Doctorado en NeurocienciasPattern recognition of neuronal discharges is the electrophysiological basis of the functional characterization of brain processes, so the implementation of a Spike Sorting algorithm is an essential step for the analysis of neural codes and neural interactions in a network or brain circuit. Extracted information from the neural action potential can be used to characterize neural activity events and correlate them during behavioral and cognitive processes, including different types of associative learning tasks. In particular, feature extraction is a critical step in the spike sorting procedure, which is prior to the clustering step and subsequent to the spike detection-identification step in a Spike Sorting algorithm. In the present doctoral thesis, the implementation of an automatic and unsupervised computational algorithm, called 'Unsupervised Automatic Algorithm', is proposed for the detection, identification and classification of the neural action potentials distributed across the electrophysiological recordings; and for clustering of these potentials in function of the shape, phase and distribution features, which are extracted from the first-order derivative of the potentials under study. For this, an efficient and unsupervised clustering method was developed, which integrate the K-means method with two clustering measures (validity and error indices) to verify both the cohesion-dispersion among neural spike during classification and the misclassification of clustering, respectively. In additions, this algorithm was implemented in a customized spike sorting software called VISSOR (Viability of Integrated Spike Sorting of Real Recordings). On the other hand, a supervised grouping method of neural activity profiles was performed to allow the recognition of specific patterns of neural discharges. Validity and effectiveness of these methods and algorithms were tested in this doctoral thesis by the classification of the detected action potentials from extracellular recordings of the rostro-medial prefrontal cortex of rabbits during the classical eyelid conditioning. After comparing the spike-sorting methods/algorithms proposed in this work with other methods also based on feature extraction of the action potentials, it was observed that this one had a better performance during the classification. That is, the methods/algorithms proposed here allowed obtaining: (1) the optimal number of clusters of neuronal spikes (according to the criterion of the maximum value of the cohesion-dispersion index) and (2) the optimal clustering of these spike-events (according to the criterion of the minimum value of the error index). The analytical implication of these results was that the feature extraction based on the shape, phase and distribution features of the action potential, together with the application of an alternative method of unsupervised classification with validity and error indices; guaranteed an efficient classification of neural events, especially for those detected from extracellular or multi-unitary recordings. Rabbits were conditioned with a delay paradigm consisting of a tone as conditioned stimulus. The conditioned stimulus started 50, 250, 500, 1000, or 2000 ms before and co-terminated with an air puff directed at the cornea as unconditioned stimulus. The results obtained indicated that the firing rate of each recorded neuron presented a single peak of activity with a frequency dependent on the inter-stimulus interval (i.e., Âż 12 Hz for 250 ms, Âż 6 Hz for 500 ms, and Âż 3 Hz for 1000 ms). Interestingly, the recorded neurons from the rostro-medial prefrontal cortex presented their dominant firing peaks at three precise times evenly distributed with respect to conditioned stimulus start, and also depending on the duration of the inter-stimulus interval (only for intervals of 250, 500, and 1000 ms). No significant neural responses were recorded at very short (50 ms) or long (2000 ms) conditioned stimulus-unconditioned stimulus time intervals. Furthermore, the eyelid movements were recorded with the magnetic search coil technique and the electromyographic (EMG) activity of the orbicularis oculi muscle. Reflex and conditioned eyelid responses presented a dominant oscillatory frequency of Âż 12 Hz. The experimental implication of these results is that the recorded neurons from the rostro-medial prefrontal cortex seem not to encode the oscillatory properties characterizing conditioned eyelid responses in rabbits. As a general experimental conclusion, it could be said that rostro-medial prefrontal cortex neurons are probably involved in the determination of CS-US intervals of an intermediate range (250-1000 ms).Universidad Pablo de Olavide. Departamento de FisiologĂ­a, AnatomĂ­a y BiologĂ­a CelularPostprin

    Brainstem plasticity in vestibular motion-processing sensorimotor networks

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    The role of the amygdala in emotional memories:a multidisciplinary approach

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    This thesis investigates the role of the amygdala for the establishment of fear memories with a multidisciplinary approach, including behavioural, psychopharmacological, genetic, molecular, and electrophysiological techniques in rats or mice, under healthy or pathological conditions. This research program aims to shed light on the acquisition and storage of emotional memories in the amygdala and closely interconnected brain areas. In one line of experiments, the molecular mechanisms leading to the establishment of fear memory traces in the amygdala were investigated. For this purpose, the functional role of the polysialylated neural cell adhesion molecule PSA-NCAM, expressed in the synaptic junction, was assessed in the amygdala – and also prefrontal cortex and hippocampus – with psychopharmacological and genetic approaches and tasks that strongly rely on these brain areas. Two lines of studies were followed: 1) amygdala-targeted cleavage and enhancement of PSA-NCAM in rats and 2) general cleavage of PSA-NCAM throughout the brain using genetically modified mice. Taken together, both approaches show that amygdaloid PSA-NCAM plays no role in the acquisition and storage of fear memories, but is rather involved in their extinction. Furthermore, the results confirm the importance of PSA-NCAM in hippocampus mediated learning and for the first time show that prefrontal cortex mediated learning depends on PSA-NCAM. These results suggest that PSA-NCAM is selectively involved in some, but not all, synaptic plasticity processes in the brain. In another line of experiments, the valproic acid (VPA) animal model of autism was used to investigate a possible contribution of the amygdala towards the autistic pathology. VPA was injected once at a specific time point during gestation, the time of neural tube closure. The offspring of such treated rats were first characterized in a broad set of behavioural tasks. It was found that VPA-treated offspring exhibited very specific behavioural anomalies closely resembling autistic symptomotology, such as impaired social interaction, exploration and recognition, enhanced repetitive behaviours, impaired sensorimotor gating and increased anxiety, while other behavioural parameters were left unharmed. Once the validity of the model was established, amygdala functionality was assessed. The results demonstrated that VPA-treated offspring exhibited highly enhanced conditioned fear memories, which generalized to other stimuli and were resistant to extinction. Electrophysiological in vitro recordings in the amygdala revealed hyper-reactivity towards stimulation and enhanced activity-induced synaptic plasticity. These results imply that enhanced activity and plasticity in the amygdala may underlie the exaggerated fear memories. Furthermore it is suggested in this thesis that a hyper-reactive amygdala may underlie some of the most basic symptoms observed in autism: reduced social interactions and resistance to rehabilitation

    Investigating human Schwann cell phenotypes and outcome measures of muscle reinnervation

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    Peripheral Nerve Injury (PNI) often causes partial or complete paralysis and/or loss of sensation of the segment of the body involved. Traumatic PNI is a global problem and can result in significant disability and socio-economic impacts. Clinical translation of new therapeutics for the treatment of PNI is challenged by the little information that is known about the cellular and molecular features that underpin human nerve regeneration. Moreover, clinical models and measurements that can quantify the efficacy of new treatments for PNI are not well established. Therefore, this PhD explored injured and healthy human nerve samples liberated from reconstructive nerve procedures to characterise the cellular and molecular features of human peripheral nerve degeneration. Associated with this theme of characterisation of human nerve injury, the recovery of motor units in reinnervated elbow flexor muscles following nerve transfer was quantified using Motor Unit Number Estimation (MUNE). In order to better understand the relationship of MUNE with the biological process of nerve regeneration, an animal model of nerve injury was used to investigate the association between MUNE and histological markers of regeneration. MUNE was found to be a sensitive marker of muscle reinnervation in human and animal models of nerve regeneration. Moreover, MUNE demonstrated a correlation with histological markers of muscle reinnervation. It is known that these changes in the number of motor units are accompanied by changes in muscle volume. Therefore, using the same surgical scenario of nerve transfer to reanimate elbow flexor muscles, this PhD measured the recovery of muscle volume following nerve transfer to reanimate elbow flexor muscles using quantitative Magnetic Resonance Imaging (MRI) techniques. It was found that MRI assessment of muscle volume is a measure that is sensitive to the biological process of nerve regeneration. With further data, this has the capacity to determine the efficacy of new therapeutics for the treatment of PNI and predict the likely functional recovery following PNI. In summary, the findings represent an important step towards understanding the in vivo cellular and molecular events in human nerve degeneration. In addition, MUNE and quantitative MRI techniques were found to represent sensitive and responsive measures of nerve regeneration. With further data, the findings presented here will help new therapeutic options for human nerve injury advance

    Electrical stimulation and activity for axonal regeneration

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    To date, there are no specific treatments available that efficiently target the loss of neural connectivity after a spinal cord injury (SCI). Thus patients usually suffer from life-long motor, sensory and autonomic dysfunction. Neuron-intrinsic growth programs are activated after a lesion in the peripheral nervous system (PNS) and can contribute to enhanced regeneration in a subsequent central lesion. Yet this so-called conditioning lesion (CL) holds little translational potential for SCI. Electrical stimulation (ES) can influence various cellular functions, including neuronal growth and could provide a practical approach to enhance regeneration after SCI. However, the mechanisms and a practical means for applying ES as a therapy after SCI are insufficiently understood. I hypothesized that evoked neuronal activity by direct ES of the peripheral nerve can enhance the growth potential of dorsal root ganglia (DRG) neurons in a similar way to CL, supporting the regeneration of the injured central branch ascending in the dorsal column. ES (20Hz, 2*MT, 0.2ms, 1h) was applied in vivo to the sciatic nerve of adult Fischer 344 rats, followed by ex-vivo assessment of the growth potential, showing about 2-fold enhanced neurite growth compared to sham animals. ES increased the percentage of neurons with neurites >100um, but there was no change in the percentage of neurite bearing neurons, indicating that the effect on growth is due to enhanced elongation and not initiation. Longer duration stimulation (7h) also enhances growth by 67 ± 25%, as well as repeated stimulation for 7 days (55 ± 24%). The pattern of growth and timeline is similar to a CL, suggesting a similar or a partial overlap in the mechanism. Growth effects of 1h ES were also assessed in vivo in a model of spinal cord injury, together with cell transplantation of BMSCs (bone marrow stromal cells) at 4 weeks post-injury. Stimulated fibers were labeled by sciatic nerve injection of the transganglionic tracer Cholera toxin B (CTB). Animals with ES for 1h showed significantly increased axonal regeneration into the spinal cell graft within the lesion compared to sham animals. Repeated stimulation with chronic electrodes showed a similar effect, but also a slight influence from chronic electrode implantation in chronic sham animals. Dieback of axons was not modified in any of the conditions. To evaluate possible side effects that may interfere with clinical applicability, I also tested pain-like behavior, showing a lack of allodynia or thermal hyperalgesia after ES. This further highlights the translational potential of this strategy in combinatorial approaches such as cell transplantation. In parallel, I investigated the mechanisms underlying the observed neuronal activity-mediated increases in neurite growth. Using in vitro depolarization of DRG neurons as a model, my data show that neurite growth is influenced depending on the duration of the depolarization and the delay between stimulation and measurement. Since depolarization induces calcium influx, I examined in a separate set of experiments calcium signaling, showing that blocking nuclear calcium signaling with recombinant calmodulin-binding proteins reduces growth in DRG cultures at 72h by 50 ± 10%. However, a cytoplasmic block enhances growth by 35 ± 11%, and has similar effects in vivo after adeno-associated virus gene transfer into lumbar DRGs. This differential effect of nuclear and cytoplasmic calcium signaling provides an explanation for previous reports, which have shown stimulation or reduction of growth following neuronal activity. Furthermore, I investigated HDAC5 (histone deacetylase 5), showing export from the nucleus in DRGs (92 ± 5% nuclear before and 14 ± 1% after depolarization). These in vitro experiments suggest that neuronal activity-mediated effects on axon growth could involve epigenetic mechanisms, dependent on calcium/calmodulin signaling. To follow up on these experiments, RNA sequencing was performed to investigate differential gene expression at 1 day and 7 days after ES, compared to sham animals, naive animals and animals that underwent a peripheral lesion, collecting 30M SE reads/sample on a HiSeq2000. As expected CL induces and represses an extensive number of genes compared to naïve animals. ES induced/reduced expression of a much lower number of genes relative to sham animals with smaller changes in gene expression. Several genes and pathways could be identified that are known to play a role in regeneration, suggesting that ES-mediated effects on axon regeneration are likely a summation of several activated pathways that overlap only partially with CL. Taken together, my results reveal the capacity of neurons to modulate their growth response depending on their activity in vivo. Electrical stimulation is shown to be an effective means to increase axonal regeneration in a central lesion, and could provide a feasible therapeutic approach either alone or in combination with other strategies such as cell transplantation

    Spike sorting paradigm for classification of multi-channel recorded fasciculation potentials

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    Objective There is growing interest in the relationship between sedentary behaviour and mental distress among adolescents, but the majority of studies to date have relied on self-reported measures with poor validity. Consequently, current knowledge may be affected by various biases. The aim of this study was to investigate the cross-sectional and longitudinal association between (1) objectively measured sedentary time and (2) self-reported screen time with mental distress among adolescents participating in The Tromsø Study: Fit Futures, in order to see if the association is dependent on mode of measurement of sedentary behaviour. Design Prospective study. Setting Sample drawn from upper secondary school students (mean age 16.3 years at baseline) from two municipalities in Northern Norway participating in The Tromsø Study: Fit Futures 1 and 2. Participants 686 adolescents (54.5% female), with complete self-reported and accelerometer data after multiple imputation. Primary outcome measures Mental distress assessed via the Hopkins Symptom Checklist-10 (HSCL-10). Results Minutes in sedentary behaviour measured by accelerometer showed no significant relationship with mental distress in neither crude, partly adjusted nor multiple adjusted hierarchic linear regression analyses. Self-reported screen time was positively associated with mental distress in all analyses (multiple adjusted, B=0.038, p=0.008, 95% CI 0.010 to 0.066). However, the effect was small. Conclusions Self-reported screen time was associated with slightly elevated mental distress 2 years later, whereas objectively measured minutes in sedentary behaviour was not, indicating a discrepancy in the results depending on measurement methods
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