Modulation of in Vivo Neural Network Activity with Electrochemically Controlled Delivery of Neuroactive Molecules

Neural interface technologies with implantable microelectrode arrays hold great promise for treating neural injuries or disorders. On neural electrode surfaces, conducting polymers can be electropolymerization with negatively charged molecules incorporated. When the polymer is reduced with negative current, dopant molecules are released from the polymer. This feature can be utilized to deliver neural transmitters and modulators from the electrodes to alter neural network activity. Previously, release of CNQX (6-cyano-7-nitroquinoxaline-2,3-dione), an AMPA (2-amino-3-(5-methyl-3-oxo-1,2- oxazol-4-yl)propanoic acid) receptor antagonist in hippocampal neuron culture effectively suppressed local neural activity in a transient manner. In this study, we further advance this technology by characterizing the drug loading and release capacity from microelectrodes, expanding the range of candidate dopants, and demonstrating in vivo effectiveness in rat somatosensory (S1) barrel cortex. Firstly, to quantify the concentration of released drug, fluorescent model molecule was used and quantitatively assessed in a real time imaging system. Stimulation amplitude was varied to determine the amount of released drug from microelectrodes. Secondly, only negatively charged drugs have been effectively released in the past. In this study, zwitterionic transmitter γ-Aminobutyric acid (GABA) was successfully delivered with the technique, greatly expanding the applicable range for the technique. Finally, we used evoked response from barrel cortex to evaluate the release of DNQX (6,7-dinitroquinoxaline-2,3-dione), an analog of CNQX. The neural activity of barrel cortex reliably represents sensory stimuli from whiskers, hence provides an excellent in vivo network model for evaluating our neurochemical release system. Neural activity from multi-whisker stimulation was immediately and locally suppressed by released DNQX for one to six seconds, demonstrating the high spatial-temporal resolution of the technique. Furthermore, weaker activities were nearly abolished by released DNQX whilst stronger activities were less influenced, because the strong over-saturated neural input can only be partially antagonized. The system demonstrates successful modulation of neural network activity in a highly controllable manner. With the ease of being incorporated in existing neural implants without increasing the volume or complexity, this technology may find use in a wide range of neuroscience studies and potentially therapeutic devices

Advances in nanomaterials integration in CMOS-based electrochemical sensors: a review

The monolithic integration of electrochemical sensors with instrumentation electronics on semiconductor technology is a promising approach to achieve sensor scalability, miniaturization and increased signal to noise ratio. Such an integration requires post-process modification of microchips (or wafers) fabricated in standard semiconductor technology (e.g. CMOS) to develop sensitive and selective sensing electrodes. This review focuses on the post-process fabrication techniques for addition of nanomaterials to the electrode surface, a key component in the construction of electrochemical sensors that has been widely used to achieve surface reactivity and sensitivity. Several CMOS-compatible techniques are summarized and discussed in this review for the deposition of nanomaterials such as gold, platinum, carbon nanotubes, polymers and metal oxide/nitride nanoparticles. These techniques include electroless deposition, electro-chemical deposition, lift-off, micro-spotting, dip-pen lithography, physical adsorption, self-assembly and hydrothermal methods. Finally, the review is concluded and summarized by stating the advantages and disadvantages of these deposition methods

Investigating epileptiform activity associated with slow wave sleep

PhD ThesisThe characteristic EEG trait of patients with nocturnal idiopathic epilepsies during childhood is the spike and wave discharge. Cognitive dysfunction is prevalent among these patients and is thought to be linked to disturbances in memory consolidation processes that normally occur during slow wave sleep. Several genetic mutations of nicotinic receptor subunits have been linked to these disorders. However, there is little known about the underlying mechanisms or the spatiotemporal characteristics of this epileptiform activity within the neocortex. This thesis presents a rat in vitro model of the epileptiform activity synonymous with nocturnal childhood epilepsies, that allows for pharmacological manipulation of receptor subunits linked to these disorders. The application of DTC [10 M], a non-selective, competitive nicotinic acetylcholine receptor antagonist, to an in vitro model of the cortical delta rhythm induced two individual forms of paroxysm events - wave discharges and the conventional spike and wave discharges. Pharmacological manipulation of this model suggest that the epileptiform activity is mediated by excitatory currents which is consistent with the use of glutamate antagonists as anticonvulsants. A blanket blockade of inhibition by a GABAA antagonist resulted in severe discharges, hence hugely increasing excitatory response. Only partial disinhibition is suggested to be required to generate epileptiform activity as nicotinic acetylcholine receptors and 5-HT3 receptors are located on dendrite targeting interneurons. Mapping of unit activity revealed the di erence between the two paroxysm events was recruitment of super cial layers with simultaneous paroxysm events in delta frequency-generating Layer V pyramidal cells. It is proposed that the hyperexcitability responsible for the generation of the spike component of a spike and wave discharge is mediated by the lack of excitatory tone in 5-HT3 and nicotinic acetylecholine receptor expressing inhibitory interneuron subtypes. The disinhibition, spike generation and disruption of interplay between deep and super cial layers of the neocortex is thought to be associated with synaptic plastic changes

Dynamics of serotonergic neurons revealed by fiber photometry

This work was developed in the context of the MIT Portugal Program, area of Bioengineering Systems, in collaboration with the Champalimaud Research Programme, Champalimaud Center for the Unknown, Lisbon, Portugal. The project entitled Dynamics of serotonergic neurons revealed by fiber photometry was carried out at Instituto Gulbenkian de Ciência, Oeiras, Portugal and at the Champalimaud Research Programme, Champalimaud Center for the Unknown, Lisbon, PortugalSerotonin is an important neuromodulator implicated in the regulation of many physiological and cognitive processes. It is one of the most studied neuromodulators and one of the main targets of psychoactive drugs, since its dysregulation can contribute to altered perception and pathological conditions such as depression and obsessive-compulsive disorder. However, it is still one of the most mysterious and least understood neuromodulatory systems of the brain. In order to study the activity of serotonergic neurons in behaving mice, we used genetically encoded calcium indicators and developed a fiber photometry system to monitor neural activity from genetically defined populations of neurons. This approach was developed to study serotonin neurons but it can be used in any genetically defined neuronal population. To validate our approach, we first confirmed that increased neural activity, induced by electrical microstimulation, indeed produced increases in fluorescence detected by the system. We then used it to monitor activity in the dorsal striatum of freely behaving mice. We show that the two projection pathways of the basal ganglia are both active during spontaneous contraversive turns. Additionally, we show that this balanced activity in the two pathways is needed for such contraversive movements. Finally, we used the fiber photometry system to study the role of serotonin in learning and behavioral control and to compare it to that of dopamine, another important neuromodulator. Dopamine and serotonin are thought to act jointly to orchestrate learning and behavioral control. While dopamine is thought to invigorate behavior and drive learning by signaling reward prediction errors, i.e. better-than-expected outcomes, serotonin has been implicated in behavioral inhibition and aversive processing. More specifically, serotonin has been implicated in preventing perseverative responses in changing environments. However, whether or how serotonin neurons signal such changes is not clear. To investigate these issues, we used a reversal learning task in which mice first learned to associate different odor cues with specific outcomes and then we unexpectedly reversed these associations. We show that dorsal raphe serotonin neurons, like midbrain dopamine neurons, are specifically recruited following prediction errors that occur after reversal. Yet, unlike dopamine neurons, serotonin neurons are similarly activated by surprising events that are both better and worse than expected. Dopamine and serotonin responses both track learned cue-reward associations, but serotonin neurons are slower to adapt to the changes that occur at reversal. The different dynamics of these neurons following reversal creates an imbalance that favors dopamine activity when invigoration is needed to obtain rewards and serotonin activity when behavior should be inhibited. Our data supports a model in which serotonin acts by rapidly reporting erroneous associations, expectations or priors in order to suppress behaviors driven by such errors and enhance plasticity to facilitate error correction. Contrary to prevailing views, it supports a concept of serotonin based on primary functions in prediction, control and learning rather than affect and mood

Novel Tools to Investigate Cortical Activity in Paroxysmal Disorders

This PhD project is at the interface between academic research and industry, and is jointly sponsored by the BBSRC and the industrial partner– Scientifica UK. The goal of this research is the development of new instruments and approaches to monitor and manipulate neuronal network activity in disease states. Firstly, (I) I collaborated with Scientifica to develop and utilise the newly developed Laser Applied Stimulation and Uncaging (LASU) system. The combined usage of the LASU system, alongside novel spatially-targeted channelrhodopsin variants, has al- lowed me to test the limits of single-photon optogenetic stimulation in achieving specific activation of targeted neurons. The presented findings demonstrate that, al- though high-resolution stimulation is achievable in the rodent cortex, single-photon stimulation is insufficient to achieve single-cell resolution stimulation. Secondly, (II) I have combined the high temporal resolution of novel, transparent 16-channel epicortical graphene solution-gated field effect transistor (gSGFET) arrays with the large spatial coverage of bilateral widefield Ca2+ fluorescence imaging; to per- form investigations of the relationship between spreading depolarisation (SD) and cortical seizures in awake head-fixed mouse models of epilepsy. To analyse these complex datasets, I developed a bespoke, semi-automated analysis pipeline to pro- cess the data and probe the seizure-SD relationship. I present the advantages of this dual-modality approach by demonstrating the strengths and weaknesses of each recording method, and how a synergistic approach overcomes the limitations of each technique alone. I utilise widefield imaging to perform systematic classification of SD and seizures both temporally and spatially. Detailed electrophysiological anal- ysis of gSGFET data is then performed on extracted time periods of interest. This work demonstrates the complex interaction between seizures and SD, and proposes several mechanisms describing these interactions. The technological and analytical tools presented here lay the groundwork for insightful and flexible experimental paradigms; altogether, able to probe paroxysmal activity in profound detail

NEURAL CIRCUIT DYNAMICS AND FUNCTION OF COMPLEX BEHAVIORAL STATES

Mammalian neural circuits are sophisticated biological systems that choreograph behavioral processes vital for survival. While the inherent complexity of discrete neural circuits has proven difficult to decipher, many parallel methodological developments promise to help delineate the function and connectivity of molecularly defined neural circuits. Here, I utilize novel neurotechniques to precisely monitor and manipulate anxiety- and feeding-related circuit activity. By using a holistic, multifaceted approach for perturbing and measuring neural circuit dynamics, we begin to provide a framework for understanding how adaptive and maladaptive behavioral states are manifested through the cooperative interactions of discrete extended amygdala, midbrain, and hypothalamic circuit elements.Doctor of Philosoph

Characterization of Evoked Potentials During Deep Brain Stimulation in the Thalamus

<p>Deep brain stimulation (DBS) is an established surgical therapy for movement disorders. The mechanisms of action of DBS remain unclear, and selection of stimulation parameters is a clinical challenge and can result in sub-optimal outcomes. Closed-loop DBS systems would use a feedback control signal for automatic adjustment of DBS parameters and improved therapeutic effectiveness. We hypothesized that evoked compound action potentials (ECAPs), generated by activated neurons in the vicinity of the stimulating electrode, would reveal the type and spatial extent of neural activation, as well as provide signatures of clinical effectiveness. The objective of this dissertation was to record and characterize the ECAP during DBS to determine its suitability as a feedback signal in closed-loop systems. The ECAP was investigated using computer simulation and <italic>in vivo</italic> experiments, including the first preclinical and clinical ECAP recordings made from the same DBS electrode implanted for stimulation. </p><p>First, we developed DBS-ECAP recording instrumentation to reduce the stimulus artifact and enable high fidelity measurements of the ECAP at short latency. <italic>In vitro</italic> and <italic>in vivo</italic> validation experiments demonstrated the capability of the instrumentation to suppress the stimulus artifact, increase amplifier gain, and reduce distortion of short latency ECAP signals.</p><p>Second, we characterized ECAPs measured during thalamic DBS across stimulation parameters in anesthetized cats, and determined the neural origin of the ECAP using pharmacological interventions and a computer-based biophysical model of a thalamic network. This model simulated the ECAP response generated by a population of thalamic neurons, calculated ECAPs similar to experimental recordings, and indicated the relative contribution from different types of neural elements to the composite ECAP. Signal energy of the ECAP increased with DBS amplitude or pulse width, reflecting an increased extent of activation. Shorter latency, primary ECAP phases were generated by direct excitation of neural elements, whereas longer latency, secondary phases were generated by post-synaptic activation.</p><p>Third, intraoperative studies were conducted in human subjects with thalamic DBS for tremor, and the ECAP and tremor responses were measured across stimulation parameters. ECAP recording was technically challenging due to the presence of a wide range of stimulus artifact magnitudes across subjects, and an electrical circuit equivalent model and finite element method model both suggested that glial encapsulation around the DBS electrode increased the artifact size. Nevertheless, high fidelity ECAPs were recorded from acutely and chronically implanted DBS electrodes, and the energy of ECAP phases was correlated with changes in tremor. </p><p>Fourth, we used a computational model to understand how electrode design parameters influenced neural recording. Reducing the diameter or length of recording contacts increased the magnitude of single-unit responses, led to greater spatial sensitivity, and changed the relative contribution from local cells or passing axons. The effect of diameter or contact length varied across phases of population ECAPs, but ECAP signal energy increased with greater contact spacing, due to changes in the spatial sensitivity of the contacts. In addition, the signal increased with glial encapsulation in the peri-electrode space, decreased with local edema, and was unaffected by the physical presence of the highly conductive recording contacts.</p><p>It is feasible to record ECAP signals during DBS, and the correlation between ECAP characteristics and tremor suggests that this signal could be used in closed-loop DBS. This was demonstrated by implementation in simulation of a closed-loop system, in which a proportional-integral-derivative (PID) controller automatically adjusted DBS parameters to obtain a target ECAP energy value, and modified parameters in response to disturbances. The ECAP also provided insight into neural activation during DBS, with the dominant contribution to clinical ECAPs derived from excited cerebellothalamic fibers, suggesting that activation of these fibers is critical for DBS therapy.</p>Dissertatio

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

Use of functional neuroimaging and optogenetics to explore deep brain stimulation targets for the treatment of Parkinson's disease and epilepsy

Deep brain stimulation (DBS) is a neurosurgical therapy for Parkinson’s disease and epilepsy. In DBS, an electrode is stereotactically implanted in a specific region of the brain and electrical pulses are delivered using a subcutaneous pacemaker-like stimulator. DBS-therapy has proven to effectively suppress tremor or seizures in pharmaco-resistant Parkinson’s disease and epilepsy patients respectively. It is most commonly applied in the subthalamic nucleus for Parkinson’s disease, or in the anterior thalamic nucleus for epilepsy. Despite the rapidly growing use of DBS at these classic brain structures, there are still non-responders to the treatment. This creates a need to explore other brain structures as potential DBS-targets. However, research in patients is restricted mainly because of ethical reasons. Therefore, in order to search for potential new DBS targets, animal research is indispensable. Previous animal studies of DBS-relevant circuitry largely relied on electrophysiological recordings at predefined brain areas with assumed relevance to DBS therapy. Due to their inherent regional biases, such experimental techniques prevent the identification of less recognized brain structures that might be suitable DBS targets. Therefore, functional neuroimaging techniques, such as functional Magnetic Resonance Imaging and Positron Emission Tomography, were used in this thesis because they allow to visualize and to analyze the whole brain during DBS. Additionally, optogenetics, a new technique that uses light instead of electricity, was employed to manipulate brain cells with unprecedented selectivity

Úloha Islet1, BDNF a nanočástic ve vývoji, funkci a regeneraci sluchového systému

Podmínkou úspěšného vývoje regenerační terapie ztráty sluchu je detailní znalost funkce jednotlivých genů a faktorů uplatňujících se během vývoje sluchového systému. Mezi faktory důležité pro vyvolání procesů regenerace ve vnitřním uchu patří transkripční faktor Islet1 a mozkový neurotrofní faktor BDNF. V předkládané dizertační práci je studována role obou faktorů ve vývoji a funkci sluchového systému a také možnosti využití nanočástic jako možného bezpečného prostředku pro jejich cílené doručení do kochley. U embryí transgenních myší se zvýšenou expresí Islet1 (s Pax2 promotorem) bylo pozorováno větší kochleovestibulární ganglium a indukoval se zrychlený růst a větvení nervových vláken u embryí. Funkční testy měření kmenových potenciálů a otoakustických emisí ukázaly, že u mladých transgenních myší byla sluchová funkce na úrovni kontrolních myší, ale byl pozorován brzký nástup sluchové ztráty způsobené stárnutím. Tato sluchová ztráta souvisela s degenerací zakončení eferentních kochleárních vláken mediálního olivokochleárního systému, která byla způsobena misexpresí Islet1 v zadním mozku. Tyto výsledky poprvé ukázaly, že poškození mediálního olivokochleárního systému může urychlit vznik sluchové ztráty během stárnutí bez ztráty vnějších vláskových buněk. Úloha BDNF ve sluchovém systému byla studována na...Detailed knowledge of the role that particular genes and factors play during the development and in the normal function of the auditory system is necessary to develop successful regenerative inner ear therapies. Islet1 transcription factor and brain derived neurothrophic factor (BDNF) have great potential to play a role in regenerative inner ear therapy as both have been shown to be sufficient for self-repair regeneration in cochlea in animal studies. In this study we looked at the roles these two factors play in the development and function of the auditory system. In the transgenic mice used in the study, overexpression of Isl1 affected cell specification during embryonic development, leading to enlargement of the cochleovestibular ganglion and accelerated nerve fiber extension and branching in mutant embryos. The hearing of young transgenic mice was not affected. However, it started to decline in 1-month-old animals. This early onset of age-related hearing loss was found to be a consequence of the neurodegeneration of the olivocochlear system caused by Pax2-driven Isl1 misexpression in the hindbrain. Our data provide the first evidence that the alternation of the olivocochlear system efferent system accelerates the age-related functional decline of hearing without the loss of OHCs. The functional role of...First Faculty of Medicine1. lékařská fakult