Temporal structure in spiking patterns of ganglion cells defines perceptual thresholds in rodents with subretinal prosthesis.

Subretinal prostheses are designed to restore sight in patients blinded by retinal degeneration using electrical stimulation of the inner retinal neurons. To relate retinal output to perception, we studied behavioral thresholds in blind rats with photovoltaic subretinal prostheses stimulated by full-field pulsed illumination at 20 Hz, and measured retinal ganglion cell (RGC) responses to similar stimuli ex-vivo. Behaviorally, rats exhibited startling response to changes in brightness, with an average contrast threshold of 12%, which could not be explained by changes in the average RGC spiking rate. However, RGCs exhibited millisecond-scale variations in spike timing, even when the average rate did not change significantly. At 12% temporal contrast, changes in firing patterns of prosthetic response were as significant as with 2.3% contrast steps in visible light stimulation of healthy retinas. This suggests that millisecond-scale changes in spiking patterns define perceptual thresholds of prosthetic vision. Response to the last pulse in the stimulation burst lasted longer than the steady-state response during the burst. This may be interpreted as an excitatory OFF response to prosthetic stimulation, and can explain behavioral response to decrease in illumination. Contrast enhancement of images prior to delivery to subretinal prosthesis can partially compensate for reduced contrast sensitivity of prosthetic vision

Scanned optogenetic control of mammalian somatosensory input to map input-specific behavioral outputs

Somatosensory stimuli guide and shape behavior, from immediate protective reflexes to longer-term learning and higher-order processes related to pain and touch. However, somatosensory inputs are challenging to control in awake mammals due to the diversity and nature of contact stimuli. Application of cutaneous stimuli is currently limited to relatively imprecise methods as well as subjective behavioral measures. The strategy we present here overcomes these difficulties, achieving ‘remote touch’ with spatiotemporally precise and dynamic optogenetic stimulation by projecting light to a small defined area of skin. We mapped behavioral responses in freely behaving mice with specific nociceptor and low-threshold mechanoreceptor inputs. In nociceptors, sparse recruitment of single action potentials shapes rapid protective pain-related behaviors, including coordinated head orientation and body repositioning that depend on the initial body pose. In contrast, activation of low-threshold mechanoreceptors elicited slow-onset behaviors and more subtle whole-body behaviors. The strategy can be used to define specific behavioral repertoires, examine the timing and nature of reflexes, and dissect sensory, motor, cognitive and motivational processes guiding behavior

Carbon Nanotube–Liposome Complexes in Hydrogels for Controlled Drug Delivery via Near-Infrared Laser Stimulation

Externally controllable drug delivery systems are crucial for a variety of biological applications where the dosage and timing of drug delivery need to be adjusted based on disease diagnosis and progression. Here, we have developed an externally controllable drug delivery system by combining three extensively used platforms: hydrogels, liposomes, and single-walled carbon nanotubes (SWCNTs). We have developed carbon nanotube–liposome complexes (CLCs) and incorporated these structures into a 3D alginate hydrogel for use as an optically controlled drug delivery system. The CLC structures were characterized by using a variety of imaging and spectroscopic techniques, and an optimal SWCNT/lipid ratio was selected. The optimal CLCs were loaded with a model drug (FITC-Dex), incorporated into a hydrogel, and their release profile was studied. It was shown that release of the drug cargo can be triggered by using an NIR laser stimulation tuned to the optical resonance of a particular SWCNT species. It was further shown that the amount of released cargo can be tuned by varying the NIR stimulation time. This system demonstrates the externally controlled delivery of drug cargo and can be used for different applications including cancer chemotherapy delivery

Time domain, near-infrared diffuse optical methods for path length resolved, non-invasive measurement of deep-tissue blood flow

The non-invasive and, often, continuous measurement of the hemodynamics of the body, and for the main purposes of this thesis, the brain, is desired because both the instantaneous values and their changes over time constantly adapt to the conditions affecting the body and its environment. They are altered in pathological situations and in response to increased function. It is desirable for these measurements to be continuous, reliable, minimally invasive, and relatively inexpensive. In recent years, optical techniques that, by using diffusing and deep-reaching (up to few centimeters) light at skin-safe levels of intensity, combine the aforementioned characteristics, have increasingly become used in clinical and research settings. However, to date there is, on one side the need to expand the number and scope of translational studies, and, on the other, to address shortcomings like the contamination of signals from unwanted tissue volumes (partial volume effects). A further important goal is to increase the depth of penetration of light without affecting the non-invasive nature of diffuse optics. My PhD was aimed at several aspects of this problem; (i) the development of new, more advanced methods, i.e. the time/pathlength resolved, to improve the differentiation between superficial and deeper tissues layers, (ii) the exploration of new application areas, i.e. to characterize the microvascular status of bones, to study the functional response of the baby brain, and (iii) to improve the quality control of the systems , i.e. by introducing a long shelf-life dynamic phantom. In conceptual order, first I introduce long shelf-life reference standards for diffuse correlation spectroscopy. Secondly, I describe the use of an existing hybrid time domain and diffuse correlation spectroscopy system to monitor the changes that some pathological conditions, in this case osteoporosis and human immunodeficiency virus infection, may have on many aspects of the human bone tissue that are currently not easy to measure (i.e. invasively assessed) by conventional techniques. Thirdly, I describe the development of a novel time domain optical technique that intimately combines, introducing many previously unmet advancements, the two previously cited optical spectroscopy techniques. For the first time I was able to produce a time domain device and protocol that can monitor the blood flow in vivo in the head and muscles of healthy humans. Lastly, I describe a device and method that I have used to monitor changes in blood flow in healthy human infants of three to five months of age, for the first time in this age bracket, as a marker of activation following visual stimulation. Overall, this work pushes the limit of the technology that makes use of diffuse light to minimally invasively, continuously, and reliably monitor endogenous markers of pathological and physiological processes in the human body.La medición no invasiva y, a menudo, continua de la hemodinámica del cuerpo, y para los propósitos principales de esta tesis, del cerebro, es conveniente porque tanto los valores instantáneos como sus variaciones en el tiempo se adaptan constantemente a las condiciones que afectan el cuerpo humano y su entorno. Estas suelen alterarse en situaciones patológicas o como respuesta a una mayor función. Es deseable que estas mediciones sean continuas, confiables, mínimamente invasivas y relativamente asequibles. En los últimos años, las técnicas ópticas que, mediante el uso de luz difusa para medir los tejidos en profundidad (hasta unos pocos centímetros) mediante niveles de intensidad que son seguros para la piel, combinan las características arriba mencionadas, se han utilizado cada vez más tanto en entornos clínicos como de investigación. Sin embargo, al día de hoy hay, por un lado, la necesidad de ampliar el número y el ámbito de los estudios translacionales y, por el otro, de suplir a las deficiencias como por ejemplo la contaminación de volúmenes de tejido no deseados (efectos de volumen parcial). Otro objetivo importante es aumentar la profundidad de penetración de la luz sin afectar la naturaleza no invasiva de la óptica difusa. Mi doctorado está destinado a mejorar varios aspectos de este problema; (i) el desarrollo de nuevos métodos más avanzados, es decir, el método resuelto en el tiempo/trayectoria de los fotones, para mejorar la diferenciación entre los tejidos superficiales y profundos, (ii) la exploración de nuevas áreas de aplicación, es decir, para caracterizar el estado microvascular de los huesos, para estudiar la respuesta funcional del cerebro en los niños, y (iii) para mejorar el control de calidad de los sistemas, es decir, mediante la introducción de un phantom dinámico de larga vida útil. En orden conceptual, primero voy a introducir estándares de referencia de larga vida útil para la espectroscopia de correlación difusa (DCS). En segundo lugar, voy a describir el uso de un sistema híbrido espectroscopia tiempo-resuelta (TRS) con DCS ya existente para monitorizar los cambios que algunas condiciones patológicas, en este caso la osteoporosis y la infección por el virus de la inmunodeficiencia humana, pueden comportar para muchos aspectos del tejido óseo humano que actualmente no se pueden medir con facilidad (es decir, se van evaluado de forma invasiva) mediante técnicas convencionales. En tercer lugar, voy a describir el desarrollo de una novedosa técnica óptica en el dominio temporal que combina íntimamente, introduciendo muchos avances previamente no cumplidos, TRS y DCS. Por primera vez pude producir un dispositivo y un protocolo tiempo-resueltos para medir el flujo de la sangre en la cabeza y en los músculos de seres humanos sanos. Por último, en esta tesis voy a describir un dispositivo y un método que he usado para monitorear los cambios en el flujo sanguíneo como marcadores de activación del cerebro debida a estímulos visivos en bebés entre tres y cinco meses de edad. En general, este trabajo amplia los limites de la tecnología que hace uso de la luz difusa para monitorizar, de forma mínimamente invasiva, continua y confiable los marcadores endógenos de procesos patológicos y fisiológicos en el cuerpo humano.Postprint (published version

Simultaneous mesoscopic and two-photon imaging of neuronal activity in cortical circuits.

Spontaneous and sensory-evoked activity propagates across varying spatial scales in the mammalian cortex, but technical challenges have limited conceptual links between the function of local neuronal circuits and brain-wide network dynamics. We present a method for simultaneous cellular-resolution two-photon calcium imaging of a local microcircuit and mesoscopic widefield calcium imaging of the entire cortical mantle in awake mice. Our multi-scale approach involves a microscope with an orthogonal axis design where the mesoscopic objective is oriented above the brain and the two-photon objective is oriented horizontally, with imaging performed through a microprism. We also introduce a viral transduction method for robust and widespread gene delivery in the mouse brain. These approaches allow us to identify the behavioral state-dependent functional connectivity of pyramidal neurons and vasoactive intestinal peptide-expressing interneurons with long-range cortical networks. Our imaging system provides a powerful strategy for investigating cortical architecture across a wide range of spatial scales

Hyperspectral imaging solutions for brain tissue metabolic and hemodynamic monitoring: past, current and future developments

Hyperspectral imaging (HSI) technologies have been used extensively in medical research, targeting various biological phenomena and multiple tissue types. Their high spectral resolution over a wide range of wavelengths enables acquisition of spatial information corresponding to different light-interacting biological compounds. This review focuses on the application of HSI to monitor brain tissue metabolism and hemodynamics in life sciences. Different approaches involving HSI have been investigated to assess and quantify cerebral activity, mainly focusing on: (1) mapping tissue oxygen delivery through measurement of changes in oxygenated (HbO₂) and deoxygenated (HHb) hemoglobin; and (2) the assessment of the cerebral metabolic rate of oxygen (CMRO₂) to estimate oxygen consumption by brain tissue. Finally, we introduce future perspectives of HSI of brain metabolism, including its potential use for imaging optical signals from molecules directly involved in cellular energy production. HSI solutions can provide remarkable insight in understanding cerebral tissue metabolism and oxygenation, aiding investigation on brain tissue physiological processes

Photovoltaic restoration of sight with high visual acuity

Patients with retinal degeneration lose sight due to the gradual demise of photoreceptors. Electrical stimulation of surviving retinal neurons provides an alternative route for the delivery of visual information. We demonstrate that subretinal implants with 70-μm-wide photovoltaic pixels provide highly localized stimulation of retinal neurons in rats. The electrical receptive fields recorded in retinal ganglion cells were similar in size to the natural visual receptive fields. Similarly to normal vision, the retinal response to prosthetic stimulation exhibited flicker fusion at high frequencies, adaptation to static images and nonlinear spatial summation. In rats with retinal degeneration, these photovoltaic arrays elicited retinal responses with a spatial resolution of 64 ± 11 μm, corresponding to half of the normal visual acuity in healthy rats. The ease of implantation of these wireless and modular arrays, combined with their high resolution, opens the door to the functional restoration of sight in patients blinded by retinal degeneration

Brain oscillations differentially encode noxious stimulus intensity and pain intensity

Noxious stimuli induce physiological processes which commonly translate into pain. However, under certain conditions, pain intensity can substantially dissociate from stimulus intensity, e.g. during longer-lasting pain in chronic pain syndromes. How stimulus intensity and pain intensity are differentially represented in the human brain is, however, not yet fully understood. We therefore used electroencephalography (EEG) to investigate the cerebral representation of noxious stimulus intensity and pain intensity during 10 min of painful heat stimulation in 39 healthy human participants. Time courses of objective stimulus intensity and subjective pain ratings indicated a dissociation of both measures. EEG data showed that stimulus intensity was encoded by decreases of neuronal oscillations at alpha and beta frequencies in sensorimotor areas. In contrast, pain intensity was encoded by gamma oscillations in the medial prefrontal cortex. Contrasting right versus left hand stimulation revealed that the encoding of stimulus intensity in contralateral sensorimotor areas depended on the stimulation side. In contrast, a conjunction analysis of right and left hand stimulation revealed that the encoding of pain in the medial prefrontal cortex was independent of the side of stimulation. Thus, the translation of noxious stimulus intensity into pain is associated with a change from a spatially specific representation of stimulus intensity by alpha and beta oscillations in sensorimotor areas to a spatially independent representation of pain by gamma oscillations in brain areas related to cognitive and affective-motivational processes. These findings extend the understanding of the brain mechanisms of nociception and pain and their dissociations during longer-lasting pain as a key symptom of chronic pain syndromes