The mechanisms of GABAergic signalling in the peripheral pain pathway

Peripheral pain pathway plays a crucial role in how pain is perceived and felt. The dorsal root ganglia (DRG) which house the primary sensory neurons have become the focus of many emerging pain studies due to its potential as a functional structure in controlling pain transmission, and not only for producing proteins and providing nutrients essential for neuron survival. The major inhibitory neurotransmitter in the nervous system, GABA has been shown to play a significant role in this regard. Within the present study, the mechanism of GABA release within DRG neurons was investigated by studying the expression of vesicular GABA transporter (VGAT) in the DRG neurons. VGAT was highly expressed in the DRG neuron somata. The VGAT-positive neurons also expressed markers of subpopulations of DRG neurons, including those involved in nociception. The availability of VGAT luminal (VGAT-C) and cytoplasmic (VGAT-N) domains were utilised to investigate the mechanism of GABA release in a live DRG neuron culture. This mechanism involves the recycling process of vesicles following their exocytosis. Imaging of the internalization of VGAT-C domain during vesicle recycling indicates GABA is released via exocytosis and has both, tonic and activity-dependent components. Using the in vivo electrophysiological recordings, neuronal firing in the spinal nerve and dorsal branches of the peripheral nerve (before and after the DRG, respectively), was investigated. These data revealed existence of a ‘filter’ in the DRG that decreased the frequency of the neuronal firing passing through the DRG. This filtering effect was overcome by bicuculline, a GABAA receptor antagonist indicating the role of GABAA receptor in peripheral pain pathway. This role of GABAA receptor was also supported by the decrease in GABAA receptor activation in the presence of bicuculline in DRG neurons co-cultured with HEK293 cells. In sum, in the DRG, GABA is liberated into the interneuronal space via Ca2+-dependent vesicular exocytosis, which in turn acts on GABAA receptors. This GABAergic signalling is responsible for filtering the action potentials from the periphery to the central terminals in the spinal cord. These findings identify and further characterize peripheral ‘gate’ within the somatosensory system

Conducting Polymers as Elements of Miniature Biocompatible Sensor

Conducting polymers (CPs), the so-called “fourth generation of polymeric materials”, can solve essential problems in biosensing technologies due to their unique material properties and implementation in innovative device systems. CPs have excellent biocompatibility. They can provide advantageous interfaces for bioelectrodes owing to their hybrid conducting mechanics, combining both electron and ionic charge carriers. Many (i.e. glucose) biosensors use immobilized enzymes to form a selective layer on CP structure. Miniaturization of sensors is a new requirement. Mini sensors are portable and wearable with low utilization of sample and cost-effective technology of production

Method of co-culturing mammalian muscle cells and motoneurons

The invention provides a method of co-culturing mammalian muscle cells and mammalian motoneurons. The method comprises preparing one or more carriers coated with a covalently bonded monolayer of trimethoxysilylpropyl diethylenetriamine (DETA); suspending isolated fetal mammalian skeletal muscle cells in serum-free medium according to medium composition 1; suspending isolated fetal mammalian spinal motoneurons in serum-free medium according to medium composition 1; plating the suspended muscle cells onto the one or more carriers at a predetermined density and allowing the muscle cells to attach; plating the suspended motoneurons at a predetermined density onto the one or more carriers and allowing the motoneurons to attach; covering the one or more carriers with a mixture of medium composition 1 and medium composition 2; and incubating the carriers covered in the media mixture

Neurochemical characterization of the rodent primary sensory system

Primary sensory neurons and their associated tissues are important targets for neurochemical study. Disorders of the sensory system, including chronic pain and itch, can be extremely devastating and, in many cases, difficult to treat. Part of the difficulty of treating such disorders is the limited understanding that we have for the multitude of chemical players involved in the communication of sensation within the nervous system. One particular set of intercellular signaling molecules, neuropeptides, are known to play an important role in the transmission of pain and itch signals from the peripheral system to the spinal cord. While we have a basic understanding of how many of these molecules are involved in sensory transmission, further knowledge would be benefited by more accurate and spatially relevant sampling and characterization. However, due to their low concentration and dynamic presence, the detection of these molecules in a non-targeted manner poses a unique challenge. This dissertation focuses on characterizing the peptides found in the tissues of the sensory system and released from primary sensory neurons in culture as well as improving culturing and stimulation paradigms for future research. We have worked to characterize the full content of peptides within the dorsal root ganglia, which houses the cell bodies of the primary sensory neurons, as well as other related tissues of rat and to detect changes in the peptide content of the dorsal root ganglia and dorsal horn upon generation of an itch model in mice. We have also designed a physiologically relevant sensory neuron culturing system and made strides toward spatially relevant release sampling and neuropeptide detection

Role of endocannabinoid system and acid sensing ion channels on spinal locomotor circuits during physiological and pathological conditions

Background: Mammalian spinal cord can generate well-coordinated locomotor activity called fictive locomotion in the absence of any higher brain center input or of rhythmic sensory feedback. This activity clearly provides evidence for the central pattern generator (CPG) that produces the locomotor rhythm. Such CPG consists of glutamatergic excitatory and glycinergic and GABAergic inhibitory interneuronal connections that finally excite or inhibit the motoneuronal pools. Many factors including fast (ion channels) and slow (modulatory G-protein coupled receptors; GPCRs) processes control these neuronal pools to act rhythmically. These factors are perturbed following spinal cord injury (SCI) in the early and late phases. The present study addresses the role of a few modulatory processes, namely acid sensing ion channels (ASICs) and cannabinoid 1 receptors (CB1Rs) at both initial and the late phases of injury.Objectives: Recent evidence has shown that the deletion of ASICs slows down the progression of disease in ischemic conditions, whereas the same protocol increases seizure severity. CB1R activation or deletion also results in neuroprotective or toxic mechanisms. In order to understand the importance of ASICs and CB1Rs in the spinal locomotor circuits, it is crucial to analyze them in physiological and pathological conditions. To investigate this issue, both organotypic slice culture and an in vitro rat spinal cord model were used. With the latter, fictive locomotion can be recorded from the ventral roots of the lumbar region for a time window of 24 h. Network parameters like synaptic transmission, fictive locomotion and disinhibited bursting provide information to explain the physiological modifications and pathological severity after excitotoxicity caused by transient kainate (KA; glutamate analog) application. Drugs that modulate CB1Rs and ASICs may supply evidence for the role of these processes in fictive locomotion.Results and conclusion: Our results show that the CB1R activation or block for 24 h diminished the locomotor rhythm. In particular, CB1R pharmacological block completely depressed both dorsal root (DR) and chemically evoked fictive locomotion. This depression was amplified following KA treatment. Furthermore, a limited neuroprotection was observed after CB1R agonists (anandamide; AEA or 2-arachidanoyl glycerol; 2AG) and an endogenous cannabinoid uptake inhibitor. These results allow us to propose the innate activity of CB1R (that is well preserved) to be important after KA mediated excitotoxicity, while any neuroprotective role might come in later phases after injury. A low concentration of KA that can induce a borderline injury elicited rapid glutamate release combined with proton discharge (acidification) in the organotypic SCI model. In response to this challenge, the ASIC subtypes (1a, 1b, 2a and 3) mRNA levels were found to be elevated after 24 h. Both neuronal numbers and network activity were highly depressed after application of ASIC pharmacological blockers that intensified the consequences of KA treatment. These results indicate that moderate acidification might be beneficial for the recovery (or limitation) of KA mediated excitotoxicity. Hence, this study demonstrates that both ASICs and CB1Rs activity are important in the early phase of experimental SCI in vitro. Their pharmacological modulation can outline future strategies for neuroprotection

Organic Bio-Electronics for Interfacing, Stimulating and Recording of Neural Cells

One of the main reasons for the difficulty of providing a treatment of a huge number of disorders of the brain is the lack of deeper insight on how it works. Even after centuries of research, there are no efficient tools at hand to create this deeper insight. Recently, an increasing attention is focused on organic bioelectronics, aimed at engineering, fabrication and characterization of improved biocompatible, conformable and low cost processing devices that enable stimulation and recording of neural cells to provide an unprecedented insight into the brain’s physiology and pathophysiology. These new devices are hot candidates in order to create efficient tools for research and diagnosis and to establish a working ’machine-to-brain’ communication. Still there is room for improvement in the used materials, for example in the biocompatibility. To this aim, a novel quaterthiophene (T4) semiconductor, covalently modified with lysine- ends (T4Lys), was fabricated and characterized. The material was then tested as interface for DRG primary neurons. By whole-cell patch-clamp, it was examined if the biofunctionality of neurons cultured on T4Lys was preserved and if this organic interface enabled functional differentiation of DRG primary neurons. Not only neurons but also astrocytes, their ion channels and calcium signaling play a crucial role in the physiology and pathophysiology of the central nervous system. In this context novel tools are needed to also monitor and/or modulate the astrocytic biochemical and bioelectrical activity. To this end, an organic field effect transistor device (O-CST), was interfaced with astroglial cells. In this context the biocompatibility of the organic material P13 and its preservation of the astrocytic electrophysiological properties in vitro was investigated, and the impact of a P13-based organic field effect transistor on the astrocytic ionic conductance studied. The evoked response to device stimulation was characterized and the necessity of device integrity investigated

Electronic Devices for the Combination of Electrically Controlled Drug Release, Electrostimulation, and Optogenetic Stimulation for Nerve Tissue Regeneration

[ES] La capacidad de las células madre para proliferar formando distintas células especializadas les otorga la potencialidad de servir de base para terapias efectivas para patologías cuyo tratamiento era inimaginable hasta hace apenas dos décadas. Sin embargo, esta capacidad se encuentra mediada por estímulos fisiológicos, químicos, y eléctricos, específicos y complejos, que dificultan su traslación a la rutina clínica. Por ello, las células madre representan un campo de estudio en el que se invierten amplios esfuerzos por parte de la comunidad científica. En el ámbito de la regeneración nerviosa, para modular su desarrollo y diferenciación el tratamiento farmacológico, la electroestimulación, y la estimulación optogenética son técnicas que están consiguiendo prometedores resultados. Es por ello por lo que en la presente tesis se ha desarrollado un conjunto de sistemas electrónicos para permitir la aplicación combinada de estas técnicas in vitro, con perspectiva a su aplicación in vivo. Hemos diseñado una novedosa tecnología para la liberación eléctricamente controlada de fármacos. Esta tecnología está basada en nanopartículas de sílice mesoporosa y puertas moleculares de bipiridina-heparina. Las puertas moleculares son electroquímicamente reactivas, y encierran los fármacos en el interior de las nanopartículas, liberándolos ante un estímulo eléctrico. Hemos caracterizado esta tecnología, y la hemos validado mediante la liberación controlada de rodamina en cultivos celulares de HeLa. Para la combinación de liberación controlada de fármacos y electroestimulación hemos desarrollado dispositivos que permiten aplicar los estímulos eléctricos de forma configurable desde una interfaz gráfica de usuario. Además, hemos diseñado un módulo de expansión que permite multiplexar las señales eléctricas a diferentes cultivos celulares. Además, hemos diseñado un dispositivo de estimulación optogenética. Este tipo de estimulación consiste en la modificación genética de las células para que sean sensibles a la radiación lumínica de determinada longitud de onda. En el ámbito de la regeneración de tejido mediante células precursoras neurales, es de interés poder inducir ondas de calcio, favoreciendo su diferenciación en neuronas y la formación de circuitos sinápticos. El dispositivo diseñado permite obtener imágenes en tiempo real mediante microscopía confocal de las respuestas transitorias de las células al ser irradiadas. El dispositivo se ha validado irradiando neuronas modificadas con luz pulsada de 100 ms. También hemos diseñado un dispositivo electrónico complementario de medida de irradiancia con el doble fin de permitir la calibración del equipo de irradiancia y medir la irradiancia en tiempo real durante los experimentos in vitro. Los resultados del uso de los bioactuadores en procesos complejos y dinámicos, como la regeneración de tejido nervioso, son limitados en lazo abierto. Uno de los principales aspectos analizados es el desarrollo de biosensores que permitiesen la cuantización de ciertas biomoléculas para ajustar la estimulación suministrada en tiempo real. Por ejemplo, la segregación de serotonina es una respuesta identificada en la elongación de células precursoras neurales, pero hay otras biomoléculas de interés para la implementación de un control en lazo cerrado. Entre las tecnologías en el estado del arte, los biosensores basados en transistores de efecto de campo (FET) funcionalizados con aptámeros son realmente prometedores para esta aplicación. Sin embargo, esta tecnología no permitía la medición simultánea de más de una biomolécula objetivo en un volumen reducido debido a las interferencias entre los distintos FETs, cuyos terminales se encuentran inmersos en la solución. Por ello, hemos desarrollado instrumentación electrónica capaz de medir simultáneamente varios de estos biosensores, y la hemos validado mediante la medición simultánea de pH y la detección preliminar de serotonina y glutamato.[CA] La capacitat de les cèl·lules mare per a proliferar formant diferents cèl·lules especialitzades els atorga la potencialitat de servir de base per a teràpies efectives per a patologies el tractament de les quals era inimaginable fins fa a penes dues dècades. No obstant això, aquesta capacitat es troba mediada per estímuls fisiològics, químics, i elèctrics, específics i complexos, que dificulten la seua translació a la rutina clínica. Per això, les cèl·lules mare representen un camp d'estudi en el qual s'inverteixen amplis esforços per part de la comunitat científica. En l'àmbit de la regeneració nerviosa, per a modular el seu desenvolupament i diferenciació el tractament farmacològic, l'electroestimulació, i l'estimulació optogenética són tècniques que estan aconseguint prometedors resultats. És per això que en la present tesi s'ha desenvolupat un conjunt de sistemes electrònics per a permetre l'aplicació combinada d'aquestes tècniques in vitro, amb perspectiva a la seua aplicació in vivo. Hem dissenyat una nova tecnologia per a l'alliberament elèctricament controlat de fàrmacs. Aquesta tecnologia està basada en nanopartícules de sílice mesoporosa i portes moleculars de bipiridina-heparina. Les portes moleculars són electroquímicament reactives, i tanquen els fàrmacs a l'interior de les nanopartícules, alliberant-los davant un estímul elèctric. Hem caracteritzat aquesta tecnologia, i l'hem validada mitjançant l'alliberament controlat de rodamina en cultius cel·lulars de HeLa. Per a la combinació d'alliberament controlat de fàrmacs i electroestimulació hem desenvolupat dispositius que permeten aplicar els estímuls elèctrics de manera configurable des d'una interfície gràfica d'usuari. A més, hem dissenyat un mòdul d'expansió que permet multiplexar els senyals elèctrics a diferents cultius cel·lulars. A més, hem dissenyat un dispositiu d'estimulació optogenètica. Aquest tipus d'estimulació consisteix en la modificació genètica de les cèl·lules perquè siguen sensibles a la radiació lumínica de determinada longitud d'ona. En l'àmbit de la regeneració de teixit mitjançant cèl·lules precursores neurals, és d'interés poder induir ones de calci, afavorint la seua diferenciació en neurones i la formació de circuits sinàptics. El dispositiu dissenyat permet obtindré imatges en temps real mitjançant microscòpia confocal de les respostes transitòries de les cèl·lules en ser irradiades. El dispositiu s'ha validat irradiant neurones modificades amb llum polsada de 100 ms. També hem dissenyat un dispositiu electrònic complementari de mesura d'irradiància amb el doble fi de permetre el calibratge de l'equip d'irradiància i mesurar la irradiància en temps real durant els experiments in vitro. Els resultats de l'ús dels bioactuadors en processos complexos i dinàmics, com la regeneració de teixit nerviós, són limitats en llaç obert. Un dels principals aspectes analitzats és el desenvolupament de biosensors que permeteren la quantització de certes biomolècules per a ajustar l'estimulació subministrada en temps real. Per exemple, la segregació de serotonina és una resposta identificada amb l'elongació de les cèl·lules precursores neurals, però hi ha altres biomolècules d'interés per a la implementació d'un control en llaç tancat. Entre les tecnologies en l'estat de l'art, els biosensors basats en transistors d'efecte de camp (FET) funcionalitzats amb aptàmers són realment prometedors per a aquesta aplicació. No obstant això, aquesta tecnologia no permetia el mesurament simultani de més d'una biomolècula objectiu en un volum reduït a causa de les interferències entre els diferents FETs, els terminals dels quals es troben immersos en la solució. Per això, hem desenvolupat instrumentació electrònica capaç de mesurar simultàniament diversos d'aquests biosensors i els hem validat amb mesurament simultani del pH i la detecció preliminar de serotonina i glutamat.[EN] The stem cells' ability to proliferate to form different specialized cells gives them the potential to serve as the basis for effective therapies for pathologies whose treatment was unimaginable until just two decades ago. However, this capacity is mediated by specific and complex physiological, chemical, and electrical stimuli that complicate their translation to clinical routine. For this reason, stem cells represent a field of study in which the scientific community is investing a great deal of effort. In the field of nerve regeneration, to modulate their development and differentiation, pharmacological treatment, electrostimulation, and optogenetic stimulation are techniques that are achieving promising results. For this reason, we have developed a set of electronic systems to allow the combined application of these techniques in vitro, with a view to their application in vivo. We have designed a novel technology for the electrically controlled release of drugs. This technology is based on mesoporous silica nanoparticles and bipyridine-heparin molecular gates. The molecular gates are electrochemically reactive and entrap the drugs inside the nanoparticles, releasing them upon electrical stimulus. We have characterized this technology and validated it by controlled release of rhodamine in HeLa cell cultures. For combining electrostimulation and controlled drug release we have developed devices that allow applying the different electrical stimuli in a configurable way from a graphical user interface. In addition, we have designed an expansion module that allows multiplexing electrical signals to different cell cultures. In addition, we have designed an optogenetic stimulation device. This type of stimulation consists of genetically modifying cells to make them sensitive to light radiation of a specific wavelength. In tissue regeneration using neural precursor cells, it is interesting to be able to induce calcium waves, favoring the cell differentiation into neurons and the formation of synaptic circuits. The designed device enable the obtention of real-time images through confocal microscopy of the transient responses of cells upon irradiation. The device has been validated by irradiating modified neurons with 100 ms pulsed light stimulation. We have also designed a complementary electronic irradiance measurement device to allow calibration of the irradiator equipment and measuring irradiance in real time during in vitro experiments. The results of using bioactuators in complex and dynamic processes, such as nerve tissue regeneration, are limited in an open loop. One of the main aspects analyzed is the development of biosensors that would allow quantifying of specific biomolecules to adjust the stimulation provided in real time. For instance, serotonin secretion is an identified response of neural precursor cells elongation, among other biomolecules of interest for the implementation of a closed-loop control. Among the state-of-the-art technologies, biosensors based on field effect transistors (FETs) functionalized with aptamers are promising for this application. However, this technology did not allow the simultaneous measurement of more than one target biomolecule in a small volume due to interferences between the different FETs, whose terminals are immersed in the solution. This is why we have developed electronic instrumentation capable of simultaneously measuring several of these biosensors, and we have validated it with the simultaneous pH measurement and the preliminary detection of serotonin and glutamate.Monreal Trigo, J. (2023). Electronic Devices for the Combination of Electrically Controlled Drug Release, Electrostimulation, and Optogenetic Stimulation for Nerve Tissue Regeneration [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/19384

Hope College Abstracts: 18th Annual Celebration of Undergraduate Research and Creative Activity

The 18th Annual Celebration of Undergraduate Research and Creative Activity was held on April 12, 2019 in the Richard and Helen DeVos Fieldhouse at Hope College and featured student-faculty collaborative research projects. This program is a record reflective of those projects between the 2018-2019 academic year