Two-photon imaging and analysis of neural network dynamics

The glow of a starry night sky, the smell of a freshly brewed cup of coffee or the sound of ocean waves breaking on the beach are representations of the physical world that have been created by the dynamic interactions of thousands of neurons in our brains. How the brain mediates perceptions, creates thoughts, stores memories and initiates actions remains one of the most profound puzzles in biology, if not all of science. A key to a mechanistic understanding of how the nervous system works is the ability to analyze the dynamics of neuronal networks in the living organism in the context of sensory stimulation and behaviour. Dynamic brain properties have been fairly well characterized on the microscopic level of individual neurons and on the macroscopic level of whole brain areas largely with the help of various electrophysiological techniques. However, our understanding of the mesoscopic level comprising local populations of hundreds to thousands of neurons (so called 'microcircuits') remains comparably poor. In large parts, this has been due to the technical difficulties involved in recording from large networks of neurons with single-cell spatial resolution and near- millisecond temporal resolution in the brain of living animals. In recent years, two-photon microscopy has emerged as a technique which meets many of these requirements and thus has become the method of choice for the interrogation of local neural circuits. Here, we review the state-of-research in the field of two-photon imaging of neuronal populations, covering the topics of microscope technology, suitable fluorescent indicator dyes, staining techniques, and in particular analysis techniques for extracting relevant information from the fluorescence data. We expect that functional analysis of neural networks using two-photon imaging will help to decipher fundamental operational principles of neural microcircuits.Comment: 36 pages, 4 figures, accepted for publication in Reports on Progress in Physic

Oxygen Polarography in the Awake Macaque: Bridging BOLD fMRI and Electrophysiology

Blood oxygen level dependent (BOLD) fMRI is the predominant method for evaluating human brain activity. This technique identifies brain activity by measuring blood oxygen changes associated with neural activity. Although clearly related, the nature of the relationship between BOLD fMRI identified brain activity and electrophysiologically measured neural activity remains unclear. Direct comparison of BOLD fMRI and electrophysiology has been severely limited by the technical challenges of combining the two techniques. Microelectrode electrophysiology in non-human primates is an excellent model for studying neural activity related to high order brain function similar to that commonly studied with BOLD fMRI in humans, i.e. attention, working memory, engagement. This thesis discusses the development of, validation of, and first results obtained using a new multi-site oxygen polarographic recording system in the awake macaques as a surrogate for BOLD fMRI. Oxygen polarography measures tissue oxygen which is coupled to blood oxygen. This tool offers higher resolution than BOLD fMRI and can be more readily combined with electrophysiology. Using this new tool we evaluated local field potential and oxygen responses to an engaging visual stimulus in two distinct brain systems. In area V3, a key region in the visual system and representative of stimulus driven sensory cortex, we show increased tissue oxygen and local field potential power in response to visual stimulus. In area 23 of the posterior cingulate cortex (PCC), a hub of the default-mode network we show decreased oxygen and local field potential in response to the same stimulus. The default-mode network is a set of brain regions identified in humans whose BOLD fMRI activity is higher at rest than during external engagement, arguing that they sub-serve a function that is engaged as the default-mode in humans. Our results provide new evidence of default-mode network activity in the macaque similar to that seen in humans, provide evidence that the BOLD identified default-mode suppression reflects neural suppression and overall support a strong relationship between neural activity and BOLD fMRI. However, we also note that the LFP responses in both regions show substantial nuances that cannot be seen in the oxygen response and suggest response complexity that is invisible with fMRI. Further the nature of the relationship between LFP and oxygen differs between regions. Our multi-site technique also allows us to evaluate inter-regional interaction of ongoing oxygen fluctuations. Inter-regional correlation of BOLD fMRI fluctuations is commonly used as an index of functional connectivity and has provided new insight into behaviorally relevant aspects of the brains organization and its disruption in disease. Here we demonstrate that we can measure the same inter-regional correlation using oxygen polarography. We utilize the increased resolution of our technique to investigate the frequency structure of the signals driving the correlation and find that inter-regional correlation of oxygen fluctuations appears to depend on a rhythmic mechanism operating at ~0.06 Hz

Optogenetic Interrogation and Manipulation of Vascular Blood Flow in Cortex

Understanding blood flow regulatory mechanisms that correlate the regional blood flow with the level of local neuronal activity in brain is an ongoing research. Discerning different aspects of this coupling is of substantial importance in interpretation of functional imaging results, such as functional magnetic resonance imaging (fMRI), that rely on hemodynamic recordings to detect and image brain neuronal activity. Moreover, this understanding can provide insight into blood flow disorders under different pathophysiological conditions and possible treatments for such disorders. The blood regulatory mechanisms can be studied at two different; however, complementary levels: at the cellular level or at the vascular level. To fully understand the regulatory mechanisms in brain, it is essential to discern details of the coupling mechanism in each level. While, the cellular pathways of the coupling mechanism has been studied extensively in the past few decades, our understanding of the vascular response to brain activity is fairly basic. The main objective of this dissertation is to develop proper methods and instrumentation to interrogate regional cortical vasodynamics in response to local brain stimulation. For this purpose we offer the design of a custom-made OCT scanner and the necessary lens mechanisms to integrate the OCT system, fluorescence imaging, and optogenetic stimulation technologies in a single system. The design uses off-the-shelf components for a cost-effective design. The modular design of the device allows scientists to modify it in accordance with their research needs. With this multi-modal system we are able to monitor blood flow, blood velocity, and lumen diameter of pial vessels, simultaneously. Additionally, the system design provides the possibility of generating arbitrary spatial stimulation light pattern on brain. These abilities enables researchers to capture more diverse datasets and, eventually, obtain a more comprehensive picture of the vasodynamics in the brain. Along with the device we also proposed new biological experiments that are tailored to investigate the spatio-temporal properties of the vascular response to optical neurostimulation of the excitatory neurons. We demonstrate the ability of the proposed methods to investigate the effect of length and amplitude of stimulation on the temporal pattern of response in the blood flow, blood velocity, and diameter of the pial vessels. Moreover, we offer systemic approaches to investigate the spatial characteristics of the response in a vascular network. In these methods we apply arbitrary spatial patterns of optical stimulation to the cortex of transgenic mice and monitor the attributes of surrounding vessels. With this flexibility we were able to image the brain region that is influenced by a pial artery. After characterizing the spatio-temporal properties of the vascular blood flow response to optical neuro-modulation, we demonstrate the design and application of an optogenetic-based closed-loop controller mechanism in the brain. This controller, uses a proportional–integral–derivative (PID) compensator to engineer temporal optogenetic stimulation light pulses and maintain the flow of blood at various user defined levels in a set of selected arteries. Upon tuning the gain values of the PID controller we obtained a near to critically-damped response in the blood flow of selected arterial vessels

Fast Optical Imaging of Human Brain Function

Great advancements in brain imaging during the last few decades have opened a large number of new possibilities for neuroscientists. The most dominant methodologies (electrophysiological and magnetic resonance-based methods) emphasize temporal and spatial information, respectively. However, theorizing about brain function has recently emphasized the importance of rapid (within 100 ms or so) interactions between different elements of complex neuronal networks. Fast optical imaging, and in particular the event-related optical signal (EROS, a technology that has emerged over the last 15 years) may provide descriptions of localized (to sub-cm level) brain activity with a temporal resolution of less than 100 ms. The main limitations of EROS are its limited penetration, which allows us to image cortical structures not deeper than 3 cm from the surface of the head, and its low signal-to-noise ratio. Advantages include the fact that EROS is compatible with most other imaging methods, including electrophysiological, magnetic resonance, and trans-cranial magnetic stimulation techniques, with which can be recorded concurrently. In this paper we present a summary of the research that has been conducted so far on fast optical imaging, including evidence for the possibility of recording neuronal signals with this method, the properties of the signals, and various examples of applications to the study of human cognitive neuroscience. Extant issues, controversies, and possible future developments are also discussed

Angiography and Monitoring of Hemodynamic Signals in the Brain via Optical Coherence Tomography

The brain is a complex network of interconnected neurons with each cell functioning as a nonlinear processing unit. Neural responses to stimulus can be described by activity in neurons. While blood flow changes have been associated with neural activity and are critical to brain function, this neurovascular coupling is not well understood. This work presents a technique for neurovascular interrogation, combining optogenetics and optical coherence tomography. Optogenetics is a recently developed neuromodulation technique to control activity in the brain using light with precise spatial neuronal control and high temporal resolution. Using this method, cells act as light-gated ion channels and respond to photo stimulation by increasing or decreasing activity. Spectral-domain optical coherence tomography (SD-OCT) is a noninvasive imaging modality that has the ability to image millimeter range depth and with micrometer resolution. SD-OCT has been shown to image rodent cortical microvasculature in-vivo and detect hemodynamic changes in blood vessels. Our proposed system combines optogenetics and SD-OCT to image cortical patches of the brain with the capability of simultaneously stimulating the brain. The combination allows investigation of the hemodynamic changes in response to neural stimulation. Our results detected changes in blood vessel diameter and velocity before, during and after optogenetic stimulation and is presented

Near-Infrared Spectroscopy for Brain Computer Interfacing

A brain-computer interface (BCI) gives those suffering from neuromuscular impairments a means to interact and communicate with their surrounding environment. A BCI translates physiological signals, typically electrical, detected from the brain to control an output device. A significant problem with current BCIs is the lengthy training periods involved for proficient usage, which can often lead to frustration and anxiety on the part of the user and may even lead to abandonment of the device. A more suitable and usable interface is needed to measure cognitive function more directly. In order to do this, new measurement modalities, signal acquisition and processing, and translation algorithms need to be addressed. This work implements a novel approach to BCI design, using noninvasive near-infrared spectroscopic (NIRS) techniques to develop a userfriendly optical BCI. NIRS is a practical non-invasive optical technique that can detect characteristic haemodynamic responses relating to neural activity. This thesis describes the use of NIRS to develop an accessible BCI system requiring very little user training. In harnessing the optical signal for BCI control an assessment of NIRS signal characteristics is carried out and detectable physiological effects are identified for BCI development. The investigations into various mental tasks for controlling the BCI show that motor imagery functions can be detected using NIRS. The optical BCI (OBCI) system operates in realtime characterising the occurrence of motor imagery functions, allowing users to control a switch - a “Mindswitch”. This work demonstrates the great potential of optical imaging methods for BCI development and brings to light an innovative approach to this field of research

Novel contrasts in photoacoustic tomography

Photoacoustic tomography (PAT) combines rich optical contrast and high ultrasonic resolution in optically scattering tissue at depths. Taking advantage of its 100% sensitivity to optical absorption, PAT has been widely applied to structural, functional and molecular imaging, with both endogenous and exogenous contrasts, at superior depths than pure optical methods. This dissertation explores novel absorption contrast mechanisms of PAT based on optical/thermal patterns, endogenous cellular chromophores, nanoparticles, small-molecule dyes and genetically-encoded proteins. With these novel contrasts, the proof-of-concept applications of PAT have been extended to include homogenous flow measurements, targeted angiogenesis imaging and therapy, label-free white blood cell imaging, 3D-whole-organ cell nuclei imaging with a subcellular resolution, and in vivo neural activity imaging with voltage/calcium-sensitive indicators. Specifically, Chapter 1 introduces photoacoustic microscopy (PAM) and photoacoustic computed tomography (PACT) systems and discuss the motivation of the dissertation. Chapter 2 describes two photoacoustic (PA) flow measurement methods with optical and thermal patterns, which are applicable to homogenous flowing medium. In the first method, a Doppler frequency shift in PA signals of the flow was detected and used to calculate flow speeds. In the second method, unique features in an externally imposed thermal pattern of the flow, captured by repeated B-scans along the flow direction with a PAM system, revealed different flow speeds. Chapter 3 explores the unique PA contrast of macrophages, an important type of white blood cells. Macrophages were imaged by PAM without any label, and their measured PA spectrum was distinctive from the hemoglobin spectrum, so they can be potentially differentiated from red blood cells in the blood stream. Next, with a microtomy-assisted PAM system, cell nuclei distribution in whole organs, including mouse brain and mouse lung, were imaged with subcellular resolution. Chapter 4 introduces a type of target copper nanoparticles, which are less expensive and more biocompatible than its counterpart gold nanoparticles. The PA signals of neovasculature in the mouse flank were enhanced by the ___3-targeted copper nanoparticles. Moreover, the work shows the first example of a systemically targeted antiangiogenic drug delivery with a photoacoustic contrast nanoparticle in vivo. Chapter 5 demonstrates the voltage imaging capability of PA. A voltage sensitive dye with sufficient signal change was discovered and used as a PA voltage indicator for the first time. The mechanism was characterized through both PA imaging and spectroscopic methods. Its use was explored in a mouse epilepsy model and cortical electrical stimulation model in vivo. Finally, the deep imaging potential of PA was realized by imaging the voltage response of cells under 4.5 mm thick slice of rat brain tissue using a PACT system. Chapter 6 proves the neural calcium imaging capability of PA with a genetically encoded calcium indicator. In a fly model, I ambiguously demonstrated for the first time that PA can be used to imaging neural activities in the fly brain without the interference signals from hemoglobin. In the a live-mouse-brain-slice model, I successfully demonstrated the deep imaging capability of PA for calcium imaging by imaging through a 2-mm-thick scattering medium with a PACT system