Selective stimulation and conduction block of specific nerve fibers has been a major area of research in neuroscience. The potential clinical and neurophysiological applications have warranted reliable techniques for transiently blocking conduction through nerves. High Frequency Alternating Current (HFAC) waveforms have been found to induce a reversible and repeatable block in peripheral nerves; however the effect of these waveforms on the neural activity of individual fiber types is currently unknown. Understanding this effect is critical if clinical applications are to be pursued. This dissertation work utilized extracellular electrophysiological techniques to characterize the activity of different fiber type populations in peripheral nerves during application of HFAC waveforms. First, we investigated the phenomenon in the homogeneous unmyelinated nerves of the sea-slug, Aplysia californica. Although complete reversible block was demonstrated in these nerves, a non-monotonic relationship of block threshold to frequency was found which differed from previously published work in the field. We then investigated the effect of HFAC waveforms on amphibian mixed nerves and studied the response of specific fiber types by isolating different components of the compound action potential. We validated our results from the Aplysia nerves by determining the block thresholds of the larger diameter, myelinated A-fibers and comparing them with those of the smaller diameter, unmyelinated C-fibers. We also showed that block threshold behavior during application of the HFAC waveform depends on the nerve fiber type, and this property can be used to selectively block specific fiber types. Finally, we examined the recovery time after block induction in unmyelinated nerves and found that recovery from block was dependent on the duration of application of the HFAC waveform. The time-dependent distribution of the recovery time and the non-monotonic threshold behavior in the smaller diameter unmyelinated nerves indicate that multiple mechanisms are involved in block induction using HFAC waveforms, and these mechanisms are dependent not only on the blocking stimulus but also on the characteristics of the nerve fiber. Overall, this work demonstrates that HFAC waveforms may enable inherent peripheral nerve properties to be exploited for potential clinical applications related to the treatment of unwanted neural activity.

This work presents a high-voltage, high-precision bi-directional multi-channel system capable of stimulating neural activity through bi-phasic pulses of amplitude up to ∓50 V while recording very low-voltage responses as low as tens of microvolts. Most of the systems reported from the scientific community possess at least one of the following common limitations: low stimulation voltages, low gain capabilities, or insufficient bandwidth to acquire a wide range of different neural activities. While systems can be found that present remarkable capabilities in one or more specific areas, a versatile system that performs over all these aspects is missing. Moreover, as many novel materials, like silicon carbide, are emerging as biocompatible interfaces, and more specifically as neuronal interfaces, it becomes mandatory to have a system operating across a wide range of voltages and frequencies for both physiological and electrical compatibility testing. The system designed and proven during this doctoral research effort features a ∓50 V bi-phasic pulse generator, 62 to 100 dB of software selectable amplification, and a wide 18 Hz to 12 kHz bandwidth. In addition to design and realization we report about biological testing consisting in the acquisition of neural signals from tissue cultures using an MEA where faithful signal recording was achieved with superior fidelity to a commercial system used to sample signals from the same culture. The only system parameter that was less robust than the commercial system was the noise level, which due to our higher bandwidth was somewhat expected. More importantly our custom electronics outperformed in terms of lower delay and lower cost of realization. All of these results plus suggested future works are listed for the reader's convenience.

No description available.

In this dissertation I introduce a simple experimental approach for studying afferent pain fibre physiology. I developed an en bloc dural-skull preparation that pairs electrophysiological stimulations, pharmacological manipulations, and the UV photolysis of caged compounds in and around selectively identified individual C-fibre nociceptors with microfluorometric imaging of Ca2+ responses. This allows the observation of physiological functioning in individual nociceptive fibre free nerve endings. I show high-resolution functional imaging of single action potential-evoked fluorescent transients, as well as sub- and supra-threshold calcium signaling events within individual nociceptive fibre terminations. Utilizing the dural-skull preparation I was able to identify a peripheral mechanism of action in the terminals of CGRP nociceptive fibres for an effective migraine therapeutic, the selective 5-HT1 receptor agonist, sumatriptan. I found sumatriptan to cause an approximately 40% reduction in the amplitude of action potential-evoked Ca2+ transients in the peripheral terminals of CGRP nociceptive fibres that was mediated selectively through the inhibition of N-type Ca2+ channels. Observations from this study support a peripheral site of action for sumatriptan in inhibiting the activity of dural pain fibres and adds to our understanding of the mechanisms that underlie the clinical effectiveness of 5-HT1 receptor agonists such as sumatriptan. While μ-opioid receptor agonists remain the most powerful analgesics for the treatment of severe pain, their mechanism of action in peripheral primary afferent pain fibres remain to be established. Further exploiting the dural-skull preparation I found activation of μ-opioid receptors in individual CGRP terminals had a dual modulatory effect; inhibition of N-type Ca2+ channel signaling and a frequency dependent, BKCa channel-mediated, suppression of action potential firing. These results establish possible anti-nociceptive mechanisms of μ-opioid receptor activation in the peripheral terminals of CGRP nociceptive fibres and identify new pathways to target for peripherally mediated analgesia. The development and subsequent testing of the dural-skull preparation in this dissertation displays its utility and opens up a new window for studying nociceptive fibre physiology and pathophysiology.

Submitted by Helmut Patrocinio (hell.kenn@gmail.com) on 2017-11-19T18:31:55Z No. of bitstreams: 1 Adriado_Tort_Disserta??o.pdf: 21259604 bytes, checksum: 641564a87ee862d7f3df6a7050e5f6f4 (MD5) / Approved for entry into archive by Ismael Pereira (ismael@neuro.ufrn.br) on 2017-11-22T11:58:32Z (GMT) No. of bitstreams: 1 Adriado_Tort_Disserta??o.pdf: 21259604 bytes, checksum: 641564a87ee862d7f3df6a7050e5f6f4 (MD5) / Made available in DSpace on 2017-11-22T11:59:01Z (GMT). No. of bitstreams: 1 Adriado_Tort_Disserta??o.pdf: 21259604 bytes, checksum: 641564a87ee862d7f3df6a7050e5f6f4 (MD5) Previous issue date: 2005-11-27 / A presente disserta??o, como j? indicado no pr?prio t?tulo, versa sobre exemplos de aplica??es da teoria dos processos estoc?sticos a problemas oriundos de biologia e medicina. ? ineg?vel o imenso aux?lio que a matem?tica prestou e vem prestando no desenvolvimento de conhecimentos de diversas ?reas de pesquisa ao longo da hist?ria da ci?ncia. Em particular, numa estreita rela??o simbi?tica com a f?sica, modelos matem?ticos realizaram grandes proezas ao descrever e predizer satisfatoriamente diversos fen?menos , fazendo at? mesmo com que a matem?tica fosse valorizada a um n?vel de linguagem pr?pria da natureza, principalmente no in?cio do s?culo passado com as grandes revolu??es da f?sica te?rica. Em grande parte inspirados por tal sucesso, muitos matem?ticos t?m se voltado para estudar e tentar descrever os fen?menos biol?gicos. A biologia matem?tica, que podemos dizer ser uma ci?ncia relativamente nova, ? hoje em dia um dos ramos de aplica??o da matem?tica que mais cresce, e que tem ganho cada vez mais respeito e adeptos. Principalmente a partir da metade do ?ltimo s?culo, v?rios problemas de ?reas biom?dicas t?m sido cada vez mais atacados com o aux?lio sistem?tico da matem?tica. Geralmente, os pesquisadores experimentais das ?reas biom?dicas reportam um grande n?mero de resultados e dados emp?ricos. A matem?tica entra para tentar fazer uma costura de tais resultados, de maneira a sintetiz?-los numa teoria unificadora. Como aplica??es em outras ?reas, os modelos s?o mais elegantes quando n?o s? podem descrever resultados j? conhecidos, como tamb?m s?o capazes de predizer resultados futuros. Embora ainda seja uma ?rea de garimpo e muitos modelos est?o ainda num est?gio de primeira aproxima??o, a biologia matem?tica j? mostra importantes utilidades de aplica??es reais no entendimento dos processos biol?gicos, e inclusive alguns pr?mios Nobel j? foram distribu?dos a fisiologistas que utilizaram matem?tica nas suas pesquisas.

abstract: Mechanical properties of cells are important in maintaining physiological functions of biological systems. Quantitative measurement and analysis of mechanical properties can help understand cellular mechanics and its functional relevance and discover physical biomarkers for diseases monitoring and therapeutics. This dissertation presents a work to develop optical methods for studying cell mechanics which encompasses four applications. Surface plasmon resonance microscopy based optical method has been applied to image intracellular motions and cell mechanical motion. This label-free technique enables ultrafast imaging with extremely high sensitivity in detecting cell deformation. The technique was first applied to study intracellular transportation. Organelle transportation process and displacement steps of motor protein can be tracked using this method. The second application is to study heterogeneous subcellular membrane displacement induced by membrane potential (de)polarization. The application can map the amplitude and direction of cell deformation. The electromechanical coupling of mammalian cells was also observed. The third application is for imaging electrical activity in single cells with sub-millisecond resolution. This technique can fast record actions potentials and also resolve the fast initiation and propagation of electromechanical signals within single neurons. Bright-field optical imaging approach has been applied to the mechanical wave visualization that associated with action potential in the fourth application. Neuron-to-neuron viability of membrane displacement was revealed and heterogeneous subcellular response was observed. All these works shed light on the possibility of using optical approaches to study millisecond-scale and sub-nanometer-scale mechanical motions. These studies revealed ultrafast and ultra-small mechanical motions at the cellular level, including motor protein-driven motions and electromechanical coupled motions. The observations will help understand cell mechanics and its biological functions. These optical approaches will also become powerful tools for elucidating the interplay between biological and physical functions. / Dissertation/Thesis / Doctoral Dissertation Electrical Engineering 2016

Submitted by Leonardo Cavalcante (leo.ocavalcante@gmail.com) on 2018-07-17T18:53:07Z No. of bitstreams: 1 Arquivototal.pdf: 3232624 bytes, checksum: 559aa25bbe9205467b3fdd737354219e (MD5) / Made available in DSpace on 2018-07-17T18:53:07Z (GMT). No. of bitstreams: 1 Arquivototal.pdf: 3232624 bytes, checksum: 559aa25bbe9205467b3fdd737354219e (MD5) Previous issue date: 2016-02-29 / The high incidence of pain in the general population has encouraged research about this theme. Products derived from plant species have been widely used in the pharmacological treatment of pain relief. Recent studies have reported the important role of monoterpenes, active compounds found in the essential oils of aromatic plants, having relevant analgesic and anti-inflammatory potential. The geraniol (GER) is a monoterpenic alcohol, found in >160 essential oil of plant species, especially Cymbopogon gender. In the literature consulted, several biochemical and pharmacological properties are shown of GER: antitumor, antimicrobial, antiinflammatory, antioxidant, gastric and intestinal protector, neuroprotective and antiarrhythmic. In this study was evaluated the antinociceptive activity of GER, not yet reported, by animal behavioral and electrophysiological in vitro models. Male and female adult Swiss mice were used. Initially the acute toxicity of GER was investigated by calculating the lethal dose 50 (LD50) by intraperitoneal (i.p.) (= 199.9 mg/kg) and oral (p.o.) (> 1 g/kg). In psychopharmacological screening, after the administration of single doses of GER (i.p. and p.o.), behavioral changes were observed indicating a depressant profile on the central nervous system (CNS) and/or peripheral nervous system (SNP), and relevant antinociceptive effect of geraniol. Therefore, more specific antinociceptive property evaluation tests were performed. The GER (12.5, 25 or 50 mg/kg i.p. and 50 or 200 mg/kg p.o.) decreased (p<0.001) the number of abdominal contractions induced by i.p. injection of acetic acid, when compared with the control. The opioid antagonist naloxone (5 mg/kg) administered subcutaneously (s.c.) in mice, subsequently treated with GER (25 mg/kg i.p.), did not reverse its antinociceptive activity. The GER (12.5, 25 and 50 mg/kg i.p.) reduced (p<0.001) paw licking time in the second phase (15-30 min, inflammatory phase) of the formalin test. Also, in the glutamate test was reduced (p<0.01) paw licking time when GER 50 mg/kg i.p. administered. In a subsequent step, it was investigated the effect of GER on the excitability of peripheral nerve fibers through extracellular recording in the sciatic nerve in mice. The GER presented depressant effect of the compound action potential (CAP), which was reversed after washing and recovery period. The GER blocked components of the CAP concentration-dependent manner and exposure time to the drug: 1 mM after 120 min for the first component (Aγ and Aβ fibers) and 0.6 mM after 90 min for the second (Aγ and Aδ fibers). The concentration, which induces 50% inhibition of the peak-to-peak amplitude of the PAC (IC50) for the GER was calculated, being equal to 0.48±0.04 mM. The conduction velocity was also reduced by exposure to GER from the 0.3 mM concentration, for the 1st component [46.18±2.60 m/s to 36.04±1.60 m/s; p<0.05 (n=7)] and the 2nd component [18.37±1.31 m/s to 12.71±0.56 m/s; p<0.001 (n=7)]. In conclusion, the results obtained show that GER has antinociceptive activity, mainly in pain related to inflammation. Participation of the opioid pathway in its mechanism of action is unlikely, but the modulation of glutamatergic neurotransmission in a dose-dependent manner is a possible mechanism. Its antinociceptive activity is also related to the reduction in peripheral neuronal excitability, firstly in thinner fibers Aδ, which are directly connected to the conduction pain. / A elevada incidência da dor na população em geral tem incentivado as pesquisas entorno desse tema. Produtos oriundos de espécies vegetais têm sido amplamente utilizados no tratamento farmacológico de alívio da dor. Estudos recentes têm relatado o importante papel dos monoterpenos, princípios ativos encontrados nos óleos essenciais de plantas aromáticas, tendo relevante potencial analgésico e anti-inflamatório. O geraniol (GER) é um álcool monoterpênico, encontrado no óleo essencial de >160 espécies vegetais, especialmente do gênero Cymbopogon. Na literatura consultada, pesquisas apontam várias propriedades bioquímicas e farmacológicas para o GER: antitumoral, antimicrobiana, anti-inflamatória, antioxidante, de proteção gástrica e intestinal, neuroprotetora e antiarrítmica. Neste estudo foi avaliada a atividade antinociceptiva do GER, ainda não relatada, mediante modelos animais comportamentais e eletrofisiológicos in vitro. Foram utilizados camundongos machos e fêmeas Swiss adultos. Inicialmente, foi investigada a toxicidade aguda do GER mediante cálculo da dose letal 50 (DL50) pela via intraperitoneal (i.p.) (=199,9 mg/kg) e oral (v.o.) (>1 g/kg). Na triagem psicofarmacológica, após a subministração de doses únicas de GER (i.p. e v.o.) foram observadas alterações comportamentais que indicaram perfil depressor do sistema nervoso central (SNC) e/ou periférico (SNP), e relevante efeito antinociceptivo do geraniol. Portanto, foram realizados testes comportamentais de avaliação de propriedade antinociceptiva mais específicos. O GER (12,5; 25 e 50 mg/kg i.p. e 50 ou 200 mg/kg v.o.) reduziu (p<0,001) o número de contorções abdominais induzidas por injeção i.p. de ácido acético, quando comparado com o controle. O antagonista opióide naloxona (5 mg/kg) administrado pela via subcutânea (s.c.) em camundongos, subsequentemente tratados com GER (25 mg/kg i.p.), não reverteu sua atividade antinociceptiva. O GER (12,5; 25 e 50 mg/kg i.p.) reduziu (p<0,001) o tempo de lambida da pata na segunda fase (15-30 min, fase inflamatória) do teste da formalina. Também, no teste do glutamato houve redução (p<0,01) do tempo de lambida da pata quando administrado GER 50 mg/kg i.p. Em uma etapa subsequente, investigou-se o efeito do GER sobre a excitabilidade de fibras nervosas periféricas, mediante registro extracelular em nervo ciático de camundongo. O GER apresentou efeito depressor do potencial de ação composto (PAC), o qual foi parcialmente revertido após lavagem durante o período de recuperação. O GER bloqueou as componentes do PAC, de maneira dependente da concentração e do tempo de exposição à droga: 1 mM aos 120 min para a primeira componente (fibras Aγ e Aβ) e 0,6 mM aos 90 min para a segunda (fibras Aγ e Aδ). Foi calculada para o GER, a concentração que induz 50% de inibição da amplitude pico-a-pico do PAC (CI50), sendo igual a 0,48±0,04 mM. A velocidade de condução também, foi reduzida pela exposição ao GER, a partir da concentração de 0,3 mM para a 1ª componente [46,18±2,60 m/s para 36,04±1,60 m/s; p<0,05 (n=7)] e para a 2ª componente [18,37±1,31 m/s para 12,71±0,56 m/s; p<0,001 (n=7)]. Em conclusão, os resultados obtidos mostram que o GER tem atividade antinociceptiva, principalmente na dor relacionada à inflamação. A participação da via opióide no seu mecanismo de ação é pouco provável, mas a modulação da neurotransmissão glutamatérgica de maneira dependente da dose é um mecanismo possível. Sua atividade antinociceptiva tambèm, está relacionada à redução da excitabilidade neuronal periférica, primeiramente de fibras mais finas como Aδ, ligadas diretamente à condução da dor.

Abstract This thesis is a study of the odour responses of insect olfactory (or odorant) receptor neurons (ORN) of blowfly (Calliphora vicina), mosquito (Aedes communis), fruitflies (Drosophila melanogaster and D. virilis) and large pine weevil (Hylobius abietis). A power-law dependence (similar to Stevens' law in psychophysics) was obtained for the action potential rate of ORN responses vs. odour concentration in measurements with metal microelectrodes from blowfly ORNs and an analysis system was developed for the extracellularily recorded action potentials (or nerve pulses). Odour exposure sequences were used to study action potential rates quantitatively as a function of odour concentration in air exposure. For an odour exposure sequence, a known initial amount of the odour compound in a filter paper inside a Pasteur pipette at the beginning of repeated exposures caused a gradual dilution of the odour concentration in the exposure sequence. The concentration at each exposure was calculated according to the discrete multiple headspace extraction and dilution (DMHED) method. The estimated odour concentration was assumed to obey in the method an exponential law with respect to the exposure number in the sequence. Despite that many uncontrollable parameters remain for measuring quantitatively the characteristics of the ORNs, the results obtained, e.g., sensitivity, specificity, adaptability, and the power-law realation are both biologically and technically very interesting. A time-to-voltage converter (TVC) was utilized for the response analysis in determining action potential intervals originating from a single ORN. A precision analysis of TVC was also performed. With the mosquito (Aedes communis), fruitflies (Drosophila melanogaster and D. virilis) and large pine weevil (Hylobius abietis) antennae were tested for inhibitory and excitatory effects to find out repellents and attractants. Human sweat was found to cause strong stimulus exposure in the responses of the mosquito ORNs and Neutroil® caused inhibitory responses in pine weevil ORNs, respectively. The power-law exponents for blowfly ORNs were about 0.19 in the case of 1-hexanol (HX), 0.065 in the case of 1,4-diaminobutane (14DAB) and 0.32 in the case of butyric acid (BA). The corresponding Stevens' law exponent values 0.39 and 0.33 have been reported for HX and BA, respectively, by Patte et al. (1975).

Le potentiel d'action est un des événements de signalisation majeurs du cerveau. Ce travail est dédié à l'étude de la génération du potentiel d'action, et son impact dans le potentiel extracellulaire ainsi que dans le réseau local. Pour ce faire nous avons abordé trois questions principales. Premièrement, nous nous sommes intéressés à comprendre pourquoi les potentiels d'action ont souvent un début brutal dans les enregistrements somatiques des neurones de mammifères. Nous avons montré que l'hypothèse du couplage résistive critique explique comment le potentiel d'action est initié dans le segment initial de l'axone pour fournir le 'kink' dans le soma. Deuxièmement, nous avons évalué l'impact de la position du segment initial sur le potentiel extracellulaire. De façon importante, nous démontrons que l’impact de la position du segment initial axonal dans la forme et l’amplitude du potentiel d’action dépend de la distance entre le site d’enregistrement et l’axone, et de sa position par rapport à l’axe soma-segment initial axonal.Finalement, nous avons exploré l’impact d’un seul potentiel d’action dans l’activité de réseau, car cet effet est souvent questionné. Nos montrons qu’un seul potentiel d’action d’un neurone pyramidal hippocampique peut commencer l’activité «sharp-wave ripple” qui consiste en l’activation de multiple interneurones. L’ensemble de nos résultats montre que les potentiels d’action sont des événements complexes modelés par la biochimie de le membrane neuronale et la morphologie de l’axone. De plus, ces caractéristiques neuronales modulent fortement leur impact dans le champ extracellulaire et l’activité de réseau. / The action potential is considered one of the major signaling events in the brain.Although it has been studied for years, many questions remain unanswered. The present work is dedicated to the study of action potential generation, its impact on extracellular field and local network establishment. We considered three questions: Firstly, (i) we asked why mammalian neurons often have characteristically sharp onset in the somatic recordings of action potentials. We show that the Critical Resistive Coupling Hypothesis is sufficient to explain how the action potential is initiated in the axon initial segment to provide for the ‘kink’ in the soma, while the Back propagation Hypothesis is not sufficient to explain it. Next, (ii)we asked how the placement of the axon initial segment might affect the extracellular field. We show that the impact of the axon initial segment position on the shape and amplitude ofextracellular action potential depends on the distance between the recording site andthe axon and on its position along the soma–axon initial segment axis. Finally, (iii)we inquired if a single action potential might have an effect on the network activity. Weshow that a single action potential from a single pyramidal neuron in the hippocampus can trigger sharp-wave ripple activity consisting of the firing of multiple interneurons.Altogether, our results show that action potentials are complex events shaped by the biochemistry of the neuronal membrane and morphology of the axon. In addition these features strongly modulate the neuron’s impact on the extracellular field and network activity.

Ce travail se situe dans le cadre d'un projet pluridisciplinaire, visant à utiliser un nouveau mode de stimulation des neurones sensoriels par l'infrarouge (IR) à 1875 nm. Actuellement les prothèses cochléaires et visuelles utilisent la stimulation électrique qui permet certes de visualiser des objets et de suivre une conversation mais avec une résolution qui pourrait certainement être améliorée par un autre mode de stimulation, notamment l'infrarouge. Nous avons d'abord démontré qu'une telle technique était possible dans les cellules ganglionnaires de la rétine ainsi que celles du ganglion de Scarpa (vestibule). Les réponses biologiques obtenues sous forme de variations transitoires de calcium intracellulaire et de potentiel d'action, (enregistrées par les techniques d'imagerie calcique et de patch-clamp) nous ont permis d'approfondir cette étude. En effet, de précédents travaux ont montré la faisabilité de la stimulation optique par IR des nerfs périphériques. Mais le mécanisme à l‟origine de la réponse évoquée par IR dans le tissu biologique n'a jamais été décrit jusqu'ici. Nous décrivons pour la première fois le mécanisme moléculaire qui conduit à la genèse de VVEL (variation de potentiel de membrane évoquée par laser IR). L'élément déclencheur de ce mécanisme au niveau membranaire a été révélé à l'aide d'une approche pharmacologique. Le blocage des canaux-récepteurs thermosensibles de la famille de 'Transient Receptor Potential' (Vanilloides) par le rouge de ruthénium et le RN1734, inhibe les VVELs. Nous démontrons que le mécanisme fait intervenir des canaux sodiques et calciques dépendants du voltage, dont l'activation lors d'une stimulation par l'IR est dépendante de l'ouverture des canaux thermosensibles TRPV4. / Infrared (IR) laser irradiation has been established as an appropriate stimulus for primary sensory neurons under conditions where sensory receptor cells are impaired or lost. Yet, development of clinical applications has been impeded by lack of information about the molecular mechanisms underlying the laser induced neural response. Here, we first demonstrate that retinal and vestibular ganglion cells generate biological responses evoked by mid laser irradiation. Then, we directly address this question through pharmacological characterization of the biological response evoked by mid infrared irradiation of isolated retinal and vestibular ganglion cells from rodents. Whole-cell patch-clamp recordings reveal that both voltage-gated calcium and sodium channels contribute to the laser evoked neuronal voltage variations (LEVV). In addition, selective blockade of the LEVV by micromolar concentrations of ruthenium red and RN1734 identifies thermo-sensitive TRPV4 channels as the primary effectors of the chain reaction triggered by mid infrared laser irradiation.