Two-Photon Imaging of Calcium in Virally Transfected Striate Cortical Neurons of Behaving Monkey

Two-photon scanning microscopy has advanced our understanding of neural signaling in non-mammalian species and mammals. Various developments are needed to perform two-photon scanning microscopy over prolonged periods in non-human primates performing a behavioral task. In striate cortex in two macaque monkeys, cortical neurons were transfected with a genetically encoded fluorescent calcium sensor, memTNXL, using AAV1 as a viral vector. By constructing an extremely rigid and stable apparatus holding both the two-photon scanning microscope and the monkey's head, single neurons were imaged at high magnification for prolonged periods with minimal motion artifacts for up to ten months. Structural images of single neurons were obtained at high magnification. Changes in calcium during visual stimulation were measured as the monkeys performed a fixation task. Overall, functional responses and orientation tuning curves were obtained in 18.8% of the 234 labeled and imaged neurons. This demonstrated that the two-photon scanning microscopy can be successfully obtained in behaving primates

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

Voltage imaging of waking mouse cortex reveals emergence of critical neuronal dynamics.

Complex cognitive processes require neuronal activity to be coordinated across multiple scales, ranging from local microcircuits to cortex-wide networks. However, multiscale cortical dynamics are not well understood because few experimental approaches have provided sufficient support for hypotheses involving multiscale interactions. To address these limitations, we used, in experiments involving mice, genetically encoded voltage indicator imaging, which measures cortex-wide electrical activity at high spatiotemporal resolution. Here we show that, as mice recovered from anesthesia, scale-invariant spatiotemporal patterns of neuronal activity gradually emerge. We show for the first time that this scale-invariant activity spans four orders of magnitude in awake mice. In contrast, we found that the cortical dynamics of anesthetized mice were not scale invariant. Our results bridge empirical evidence from disparate scales and support theoretical predictions that the awake cortex operates in a dynamical regime known as criticality. The criticality hypothesis predicts that small-scale cortical dynamics are governed by the same principles as those governing larger-scale dynamics. Importantly, these scale-invariant principles also optimize certain aspects of information processing. Our results suggest that during the emergence from anesthesia, criticality arises as information processing demands increase. We expect that, as measurement tools advance toward larger scales and greater resolution, the multiscale framework offered by criticality will continue to provide quantitative predictions and insight on how neurons, microcircuits, and large-scale networks are dynamically coordinated in the brain

LFP beta amplitude is predictive of mesoscopic spatio-temporal phase patterns

Beta oscillations observed in motor cortical local field potentials (LFPs) recorded on separate electrodes of a multi-electrode array have been shown to exhibit non-zero phase shifts that organize into a planar wave propagation. Here, we generalize this concept by introducing additional classes of patterns that fully describe the spatial organization of beta oscillations. During a delayed reach-to-grasp task in monkey primary motor and dorsal premotor cortices we distinguish planar, synchronized, random, circular, and radial phase patterns. We observe that specific patterns correlate with the beta amplitude (envelope). In particular, wave propagation accelerates with growing amplitude, and culminates at maximum amplitude in a synchronized pattern. Furthermore, the occurrence probability of a particular pattern is modulated with behavioral epochs: Planar waves and synchronized patterns are more present during movement preparation where beta amplitudes are large, whereas random phase patterns are dominant during movement execution where beta amplitudes are small

Multiple functions of dopamine neurons

Dopamine neurons carry phasic signals for a limited number of behavioural events. The events include, in descending order, reward, physically intense stimuli, risk and punishment. Recent neurophysiological studies have provided interesting details on these functions

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

The optogenetic revolution in cerebellar investigations

The cerebellum is most renowned for its role in sensorimotor control and coordination, but a growing number of anatomical and physiological studies are demonstrating its deep involvement in cognitive and emotional functions. Recently, the development and refinement of optogenetic techniques boosted research in the cerebellar field and, impressively, revolutionized the methodological approach and endowed the investigations with entirely new capabilities. This translated into a significant improvement in the data acquired for sensorimotor tests, allowing one to correlate single-cell activity with motor behavior to the extent of determining the role of single neuronal types and single connection pathways in controlling precise aspects of movement kinematics. These levels of specificity in correlating neuronal activity to behavior could not be achieved in the past, when electrical and pharmacological stimulations were the only available experimental tools. The application of optogenetics to the investigation of the cerebellar role in higher-order and cognitive functions, which involves a high degree of connectivity with multiple brain areas, has been even more significant. It is possible that, in this field, optogenetics has changed the game, and the number of investigations using optogenetics to study the cerebellar role in non-sensorimotor functions in awake animals is growing. The main issues addressed by these studies are the cerebellar role in epilepsy (through connections to the hippocampus and the temporal lobe), schizophrenia and cognition, working memory for decision making, and social behavior. It is also worth noting that optogenetics opened a new perspective for cerebellar neurostimulation in patients (e.g., for epilepsy treatment and stroke rehabilitation), promising unprecedented specificity in the targeted pathways that could be either activated or inhibited

Large-scale Spatiotemporal Spike Patterning Consistent with Wave Propagation in Motor Cortex

Aggregate signals in cortex are known to be spatiotemporally organized as propagating waves across the cortical surface, but it remains unclear whether the same is true for spiking activity in individual neurons. Furthermore, the functional interactions between cortical neurons are well documented but their spatial arrangement on the cortical surface has been largely ignored. Here we use a functional network analysis to demonstrate that a subset of motor cortical neurons in non-human primates spatially coordinate their spiking activity in a manner that closely matches wave propagation measured in the beta oscillatory band of the local field potential. We also demonstrate that sequential spiking of pairs of neuron contains task-relevant information that peaks when the neurons are spatially oriented along the wave axis. We hypothesize that the spatial anisotropy of spike patterning may reflect the underlying organization of motor cortex and may be a general property shared by other cortical areas

Detection power, temporal response, and spatial resolution of IRON fMRI in awake, behaving monkeys at 3 Tesla

Thesis (Ph. D.)--Harvard-MIT Division of Health Sciences and Technology, February 2007."September 2006."Includes bibliographical references.The main goal of this thesis was to systematically characterize the detection sensitivity, temporal response, and spatial resolution of IRON contrast for fMRI within the awake, behaving monkey. Understanding these issues provides insights into the physiology of the functional response to local changes in brain activity, enables researchers to optimize experimental designs, and delineates the advantages and limitations of neuroimaging within this important animal model. The injection of the iron oxide contrast agent (MION) provided a 9-fold increase in efficiency for block designs relatively to BOLD contrast. Because the hemodynamic response function acts as a low-pass filter on neural activation to attenuate the size of differential responses to alternate stimuli, this factor dropped to approximately 2 for rapidly presented stimuli. Detection efficiency for event-related stimulus designs for BOLD and IRON contrasts could be optimized using random or semi-random distributions for interstimulus intervals. Small increases in predictability could be traded for large gains in efficiency, particularly for the IRON method. A general linear model was successfully employed to describe IRON and BOLD impulse response functions. Both responses were accurately described by a bimodal exponential model with similar time constants, a fast (4.5 sec) and a slow (13.5 sec).(cont.) The slow response comprised 80% of IRON signal, and was responsible for the BOLD post-stimulus undershoot. It likely encompasses changes in post-arteriole blood volume. Optimized IRON activation maps do not show activation in draining veins or draining tissue, in contrast with BOLD contrast. To examine what happens at the level of small vessels and capillaries, we used point-image stimuli to measure IRON and BOLD point spread functions (PSF) in V1. We estimated an IRON PSF no larger than approximately 0.4 mm, and a BOLD PSF with twice the size. Severe image distortions arising from monkey's body motion outside of the field of view currently limit the achievable spatial resolution. Preliminary data suggests multi-shot EPI with navigators may be useful in improving image stability at higher resolution for IRON fMRI, which can employ short echo times to minimize phase variations, while achieving maximum efficiency by increasing the MION dose.by Francisca Maria Pais Horta Leite.Ph.D

Characterization Of Somatosensation In The Brainstem And The Development Of A Sensory Neuroprosthesis

Innovations in neuroprosthetics have restored sensorimotor function to paralysis patients and amputees. However, to date there is a lack of solutions available to adequately address the needs of spinal cord injury patients (SCI). In this dissertation we develop a novel sensor-brain interface (SBI) that delivers electric microstimulation to the cuneate nucleus (CN) to restore somatosensory feedback in patients with intact limbs. In Chapter II, we develop a fully passive liquid metal antenna using gallium-indium (GaIn) alloy injected in polydimethylsiloxane (PDM) channels to measure forces within the physiological sensitivity of a human fingertip. In Chapter III, we present the first chronic neural interface with the CN in primates to provide access to long-term unit recordings and stimulation. In Chapter IV, we demonstrate that microstimulation to the CN is detectable in a Three Alternative Force Choice Oddity task in awake behaving primates. In Chapter V, we explore the downstream effects of CN stimulation on primary somatosensory cortex, in the context of spontaneous and evoked spindles under sedation. In summary, these findings constitute a proof-of-concept for the sensory half of a bidirectional sensorimotor prosthesis in the CN