Dendritic Ca2+ action potentials in neocortical pyramidal neurons have been characterized in brai... more Dendritic Ca2+ action potentials in neocortical pyramidal neurons have been characterized in brain slices, but their presence and role in the intact neocortex remain unclear. Here we used two-photon microscopy to demonstrate Ca2+ electrogenesis in apical dendrites of deep-layer pyramidal neurons of rat barrel cortex in vivo. During whisker stimulation, complex spikes recorded intracellularly from distal dendrites and sharp waves in the electrocorticogram were accompanied by large dendritic [Ca2+ ] transients; these also occurred during bursts of action potentials recorded from somata of identified layer 5 neurons. The amplitude of the [Ca 2+] transients was largest proximal to the main bifurcation, where sodium action potentials produced little Ca2+ influx. In some cases, synaptic stimulation evoked [Ca2+] transients without a concomitant action potential burst, suggesting variable coupling between dendrite and soma.
1. The procerebral (PC) lobe of the terrestrial mollusk Limax maximus contains a highly interconn... more 1. The procerebral (PC) lobe of the terrestrial mollusk Limax maximus contains a highly interconnected network of local olfactory interneurons that receives direct axonal projections from the two pairs of noses. This olfactory processing network generates a 0.7-Hz oscillation in its local field potential (LFP) that is coherent throughout the network. The oscillating LFP is modulated by natural odorants applied to the neuroepithelium of the superior nose. 2. Two amines known to be present in the PC lobe, dopamine and serotonin, increase the frequency of the PC lobe oscillation and alter its waveform. 3. Glutamate, another putative neurotransmitter known to be present in the lobe, suppresses the PC lobe oscillation by a quisqualate-type receptor and appears to be used by one of the two classes of neurons in the PC lobe to generate the basic LFP oscillation. 4. The known activation of second messengers in Limax PC lobe by dopamine and serotonin together with their effects on the oscill...
Larval zebrafish offer the potential for large-scale optical imaging of neural activity throughou... more Larval zebrafish offer the potential for large-scale optical imaging of neural activity throughout the central nervous system; however, several barriers challenge their utility. First, ~panneuronal probe expression has to date only been demonstrated at early larval stages up to 7 days post-fertilization (dpf), precluding imaging at later time points when circuits are more mature. Second, nuclear exclusion of genetically-encoded calcium indicators (GECIs) limits the resolution of functional fluorescence signals collected during imaging. Here, we report the creation of transgenic zebrafish strains exhibiting robust, nuclearly targeted expression of GCaMP3 across the brain up to at least 14 dpf utilizing a previously described optimized Gal4-UAS system. We confirmed both nuclear targeting and functionality of the modified probe in vitro and measured its kinetics in response to action potentials (APs). We then demonstrated in vivo functionality of nuclear-localized GCaMP3 in transgenic ...
Proceedings of the National Academy of Sciences, 1990
Paramagnetic deoxyhemoglobin in venous blood is a naturally occurring contrast agent for magnetic... more Paramagnetic deoxyhemoglobin in venous blood is a naturally occurring contrast agent for magnetic resonance imaging (MRI). By accentuating the effects of this agent through the use of gradient-echo techniques in high fields, we demonstrate in vivo images of brain microvasculature with image contrast reflecting the blood oxygen level. This blood oxygenation level-dependent (BOLD) contrast follows blood oxygen changes induced by anesthetics, by insulin-induced hypoglycemia, and by inhaled gas mixtures that alter metabolic demand or blood flow. The results suggest that BOLD contrast can be used to provide in vivo real-time maps of blood oxygenation in the brain under normal physiological conditions. BOLD contrast adds an additional feature to magnetic resonance imaging and complements other techniques that are attempting to provide positron emission tomography-like measurements related to regional neural activity.
Proceedings of the National Academy of Sciences, 2000
Neocortical pyramidal neurons have extensive axonal arborizations that make thousands of synapses... more Neocortical pyramidal neurons have extensive axonal arborizations that make thousands of synapses. Action potentials can invade these arbors and cause calcium influx that is required for neurotransmitter release and excitation of postsynaptic targets. Thus, the regulation of action potential invasion in axonal branches might shape the spread of excitation in cortical neural networks. To measure the reliability and extent of action potential invasion into axonal arbors, we have used two-photon excitation laser scanning microscopy to directly image action-potential-mediated calcium influx in single varicosities of layer 2/3 pyramidal neurons in acute brain slices. Our data show that single action potentials or bursts of action potentials reliably invade axonal arbors over a range of developmental ages (postnatal 10–24 days) and temperatures (24°C-30°C). Hyperpolarizing current steps preceding action potential initiation, protocols that had previously been observed to produce failures ...
The advent of methods for optical imaging of large-scale neural activity at cellular resolution i... more The advent of methods for optical imaging of large-scale neural activity at cellular resolution in behaving animals presents the problem of identifying behavior-encoding cells within the resulting image time series. Rapid and precise identification of cells with particular neural encoding would facilitate targeted activity measurements and perturbations useful in characterizing the operating principles of neural circuits. Here we report a regression-based approach to semiautomatically identify neurons that is based on the correlation of fluorescence time series with quantitative measurements of behavior. The approach is illustrated with a novel preparation allowing synchronous eye tracking and two-photon laser scanning fluorescence imaging of calcium changes in populations of hindbrain neurons during spontaneous eye movement in the larval zebrafish. Putative velocity-to-position oculomotor integrator neurons were identified that showed a broad spatial distribution and diversity of e...
Multiphoton excitation through optical fibers is limited by pulse broadening caused by self-phase... more Multiphoton excitation through optical fibers is limited by pulse broadening caused by self-phase modulation. We show that for short fiber lengths ͑approximately 2 m͒ two-photon excitation efficiency at the fiber output can be substantially improved by single-mode propagation in a large-area multimode fiber ͑10-m core diameter͒ instead of a standard 5.5-m core fiber. Measurements and numerical simulations of postfiber spectra and pulse widths demonstrate that the increase in efficiency is due to a reduction of nonlinear pulse broadening. Single-mode propagation in a large-core fiber is thus suitable for multiphoton applications for which pulse recompression is not possible at the fiber end.
1. The procerebral (PC) lobe of the terrestrial mollusk Limax maximus contains a highly interconn... more 1. The procerebral (PC) lobe of the terrestrial mollusk Limax maximus contains a highly interconnected network of local olfactory interneurons that receives ipsilateral axonal projections from superior and inferior noses. This network exhibits an approximately 0.7-Hz intrinsic oscillation in its local field potential (LFP). 2. Intracellular recordings show that the lobe contains at least two classes of neurons with activity phase locked to the oscillation. Neurons in one class produce periodic bursts of spikes, followed by a period of hyperpolarization and subsequently a depolarizing afterpotential. There is a small but significant chance for a second burst to occur during the depolarizing afterpotential; this leads to a double event in the LFP. Bursting neurons constitute approximately 10% of the neurons in the lobe. 3. Neurons in the other class fire infrequently and do not produce periodic bursts of action potentials. However, they receive strong, periodic inhibitory input during...
We describe how several optimization problems can be rapidly solved by highly interconnected netw... more We describe how several optimization problems can be rapidly solved by highly interconnected networks of simple analog processors. Analog-to-digital (A/D) conversion was considered as a simple optimization problem, and an A/D converter of novel architecture was designed. A/D conversion is a simple example of a more general class of signal-decision problems which we show could also be solved by appropriately constructed networks. Circuits to solve these problems were designed using general principles which result from an understanding of the basic collective computational properties of a specific class of analog-processor networks. We also show that a network which solves linear programming problems can be understood from the same concepts.
Philosophical transactions of the Royal Society of London. Series B, Biological sciences, Jan 19, 2015
The evolution of the field of neuroscience has been propelled by the advent of novel technologica... more The evolution of the field of neuroscience has been propelled by the advent of novel technological capabilities, and the pace at which these capabilities are being developed has accelerated dramatically in the past decade. Capitalizing on this momentum, the United States launched the Brain Research through Advancing Innovative Neurotechnologies (BRAIN) Initiative to develop and apply new tools and technologies for revolutionizing our understanding of the brain. In this article, we review the scientific vision for this initiative set forth by the National Institutes of Health and discuss its implications for the future of neuroscience research. Particular emphasis is given to its potential impact on the mapping and study of neural circuits, and how this knowledge will transform our understanding of the complexity of the human brain and its diverse array of behaviours, perceptions, thoughts and emotions.
Virtual reality (VR) enables precise control of an animal's environment and otherwise impossi... more Virtual reality (VR) enables precise control of an animal's environment and otherwise impossible experimental manipulations. Neural activity in rodents has been studied on virtual 1D tracks. However, 2D navigation imposes additional requirements, such as the processing of head direction and environment boundaries, and it is unknown whether the neural circuits underlying 2D representations can be sufficiently engaged in VR. We implemented a VR setup for rats, including software and large-scale electrophysiology, that supports 2D navigation by allowing rotation and walking in any direction. The entorhinal-hippocampal circuit, including place, head direction, and grid cells, showed 2D activity patterns similar to those in the real world. Furthermore, border cells were observed, and hippocampal remapping was driven by environment shape, suggesting functional processing of virtual boundaries. These results illustrate that 2D spatial representations can be engaged by visual and rotati...
To investigate the mechanisms of persistent neural activity, we obtained in vivo intracellular re... more To investigate the mechanisms of persistent neural activity, we obtained in vivo intracellular recordings from neurons in an oculomotor neural integrator of the goldfish during spontaneous saccades and fixations. Persistent changes in firing rate following saccades were associated with step changes in interspike membrane potential that were correlated with changes in eye position. Perturbation of persistent activity with brief intracellular current pulses designed to mimic saccadic input only induced transient changes of firing rate and membrane potential. When neurons were hyperpolarized below action potential threshold, position-correlated step changes in membrane potential remained. Membrane potential fluctuations were greater during more depolarized steps. These results suggest that sustained changes in firing rate are supported not by either membrane multistability or changes in pacemaker currents, but rather by persistent changes in the rate or amplitude of synaptic inputs.
In layer 2/3 pyramidal neurons of barrel cortex in vivo, calcium ion concentration ([Ca2+]) trans... more In layer 2/3 pyramidal neurons of barrel cortex in vivo, calcium ion concentration ([Ca2+]) transients in apical dendrites evoked by sodium action potentials are limited to regions close to the soma. To study the mechanisms underlying this restricted pattern of calcium influx, we combined two-photon imaging of dendritic [Ca2+] dynamics with dendritic membrane potential measurements. We found that sodium action potentials attenuated and broadened rapidly with distance from the soma. However, dendrites of layer 2/3 cells were electrically excitable, and direct current injections could evoke large [Ca2+] transients. The restricted pattern of dendritic [Ca2+] transients is therefore due to a failure of sodium action-potential propagation into dendrites. Also, stimulating subcortical activating systems by tail pinch can enhance dendritic [Ca2+] influx induced by a sensory stimulus by increasing cellular excitability, consistent with the importance of these systems in plasticity and learn...
Dendritic Ca2+ action potentials in neocortical pyramidal neurons have been characterized in brai... more Dendritic Ca2+ action potentials in neocortical pyramidal neurons have been characterized in brain slices, but their presence and role in the intact neocortex remain unclear. Here we used two-photon microscopy to demonstrate Ca2+ electrogenesis in apical dendrites of deep-layer pyramidal neurons of rat barrel cortex in vivo. During whisker stimulation, complex spikes recorded intracellularly from distal dendrites and sharp waves in the electrocorticogram were accompanied by large dendritic [Ca2+ ] transients; these also occurred during bursts of action potentials recorded from somata of identified layer 5 neurons. The amplitude of the [Ca 2+] transients was largest proximal to the main bifurcation, where sodium action potentials produced little Ca2+ influx. In some cases, synaptic stimulation evoked [Ca2+] transients without a concomitant action potential burst, suggesting variable coupling between dendrite and soma.
1. The procerebral (PC) lobe of the terrestrial mollusk Limax maximus contains a highly interconn... more 1. The procerebral (PC) lobe of the terrestrial mollusk Limax maximus contains a highly interconnected network of local olfactory interneurons that receives direct axonal projections from the two pairs of noses. This olfactory processing network generates a 0.7-Hz oscillation in its local field potential (LFP) that is coherent throughout the network. The oscillating LFP is modulated by natural odorants applied to the neuroepithelium of the superior nose. 2. Two amines known to be present in the PC lobe, dopamine and serotonin, increase the frequency of the PC lobe oscillation and alter its waveform. 3. Glutamate, another putative neurotransmitter known to be present in the lobe, suppresses the PC lobe oscillation by a quisqualate-type receptor and appears to be used by one of the two classes of neurons in the PC lobe to generate the basic LFP oscillation. 4. The known activation of second messengers in Limax PC lobe by dopamine and serotonin together with their effects on the oscill...
Larval zebrafish offer the potential for large-scale optical imaging of neural activity throughou... more Larval zebrafish offer the potential for large-scale optical imaging of neural activity throughout the central nervous system; however, several barriers challenge their utility. First, ~panneuronal probe expression has to date only been demonstrated at early larval stages up to 7 days post-fertilization (dpf), precluding imaging at later time points when circuits are more mature. Second, nuclear exclusion of genetically-encoded calcium indicators (GECIs) limits the resolution of functional fluorescence signals collected during imaging. Here, we report the creation of transgenic zebrafish strains exhibiting robust, nuclearly targeted expression of GCaMP3 across the brain up to at least 14 dpf utilizing a previously described optimized Gal4-UAS system. We confirmed both nuclear targeting and functionality of the modified probe in vitro and measured its kinetics in response to action potentials (APs). We then demonstrated in vivo functionality of nuclear-localized GCaMP3 in transgenic ...
Proceedings of the National Academy of Sciences, 1990
Paramagnetic deoxyhemoglobin in venous blood is a naturally occurring contrast agent for magnetic... more Paramagnetic deoxyhemoglobin in venous blood is a naturally occurring contrast agent for magnetic resonance imaging (MRI). By accentuating the effects of this agent through the use of gradient-echo techniques in high fields, we demonstrate in vivo images of brain microvasculature with image contrast reflecting the blood oxygen level. This blood oxygenation level-dependent (BOLD) contrast follows blood oxygen changes induced by anesthetics, by insulin-induced hypoglycemia, and by inhaled gas mixtures that alter metabolic demand or blood flow. The results suggest that BOLD contrast can be used to provide in vivo real-time maps of blood oxygenation in the brain under normal physiological conditions. BOLD contrast adds an additional feature to magnetic resonance imaging and complements other techniques that are attempting to provide positron emission tomography-like measurements related to regional neural activity.
Proceedings of the National Academy of Sciences, 2000
Neocortical pyramidal neurons have extensive axonal arborizations that make thousands of synapses... more Neocortical pyramidal neurons have extensive axonal arborizations that make thousands of synapses. Action potentials can invade these arbors and cause calcium influx that is required for neurotransmitter release and excitation of postsynaptic targets. Thus, the regulation of action potential invasion in axonal branches might shape the spread of excitation in cortical neural networks. To measure the reliability and extent of action potential invasion into axonal arbors, we have used two-photon excitation laser scanning microscopy to directly image action-potential-mediated calcium influx in single varicosities of layer 2/3 pyramidal neurons in acute brain slices. Our data show that single action potentials or bursts of action potentials reliably invade axonal arbors over a range of developmental ages (postnatal 10–24 days) and temperatures (24°C-30°C). Hyperpolarizing current steps preceding action potential initiation, protocols that had previously been observed to produce failures ...
The advent of methods for optical imaging of large-scale neural activity at cellular resolution i... more The advent of methods for optical imaging of large-scale neural activity at cellular resolution in behaving animals presents the problem of identifying behavior-encoding cells within the resulting image time series. Rapid and precise identification of cells with particular neural encoding would facilitate targeted activity measurements and perturbations useful in characterizing the operating principles of neural circuits. Here we report a regression-based approach to semiautomatically identify neurons that is based on the correlation of fluorescence time series with quantitative measurements of behavior. The approach is illustrated with a novel preparation allowing synchronous eye tracking and two-photon laser scanning fluorescence imaging of calcium changes in populations of hindbrain neurons during spontaneous eye movement in the larval zebrafish. Putative velocity-to-position oculomotor integrator neurons were identified that showed a broad spatial distribution and diversity of e...
Multiphoton excitation through optical fibers is limited by pulse broadening caused by self-phase... more Multiphoton excitation through optical fibers is limited by pulse broadening caused by self-phase modulation. We show that for short fiber lengths ͑approximately 2 m͒ two-photon excitation efficiency at the fiber output can be substantially improved by single-mode propagation in a large-area multimode fiber ͑10-m core diameter͒ instead of a standard 5.5-m core fiber. Measurements and numerical simulations of postfiber spectra and pulse widths demonstrate that the increase in efficiency is due to a reduction of nonlinear pulse broadening. Single-mode propagation in a large-core fiber is thus suitable for multiphoton applications for which pulse recompression is not possible at the fiber end.
1. The procerebral (PC) lobe of the terrestrial mollusk Limax maximus contains a highly interconn... more 1. The procerebral (PC) lobe of the terrestrial mollusk Limax maximus contains a highly interconnected network of local olfactory interneurons that receives ipsilateral axonal projections from superior and inferior noses. This network exhibits an approximately 0.7-Hz intrinsic oscillation in its local field potential (LFP). 2. Intracellular recordings show that the lobe contains at least two classes of neurons with activity phase locked to the oscillation. Neurons in one class produce periodic bursts of spikes, followed by a period of hyperpolarization and subsequently a depolarizing afterpotential. There is a small but significant chance for a second burst to occur during the depolarizing afterpotential; this leads to a double event in the LFP. Bursting neurons constitute approximately 10% of the neurons in the lobe. 3. Neurons in the other class fire infrequently and do not produce periodic bursts of action potentials. However, they receive strong, periodic inhibitory input during...
We describe how several optimization problems can be rapidly solved by highly interconnected netw... more We describe how several optimization problems can be rapidly solved by highly interconnected networks of simple analog processors. Analog-to-digital (A/D) conversion was considered as a simple optimization problem, and an A/D converter of novel architecture was designed. A/D conversion is a simple example of a more general class of signal-decision problems which we show could also be solved by appropriately constructed networks. Circuits to solve these problems were designed using general principles which result from an understanding of the basic collective computational properties of a specific class of analog-processor networks. We also show that a network which solves linear programming problems can be understood from the same concepts.
Philosophical transactions of the Royal Society of London. Series B, Biological sciences, Jan 19, 2015
The evolution of the field of neuroscience has been propelled by the advent of novel technologica... more The evolution of the field of neuroscience has been propelled by the advent of novel technological capabilities, and the pace at which these capabilities are being developed has accelerated dramatically in the past decade. Capitalizing on this momentum, the United States launched the Brain Research through Advancing Innovative Neurotechnologies (BRAIN) Initiative to develop and apply new tools and technologies for revolutionizing our understanding of the brain. In this article, we review the scientific vision for this initiative set forth by the National Institutes of Health and discuss its implications for the future of neuroscience research. Particular emphasis is given to its potential impact on the mapping and study of neural circuits, and how this knowledge will transform our understanding of the complexity of the human brain and its diverse array of behaviours, perceptions, thoughts and emotions.
Virtual reality (VR) enables precise control of an animal's environment and otherwise impossi... more Virtual reality (VR) enables precise control of an animal's environment and otherwise impossible experimental manipulations. Neural activity in rodents has been studied on virtual 1D tracks. However, 2D navigation imposes additional requirements, such as the processing of head direction and environment boundaries, and it is unknown whether the neural circuits underlying 2D representations can be sufficiently engaged in VR. We implemented a VR setup for rats, including software and large-scale electrophysiology, that supports 2D navigation by allowing rotation and walking in any direction. The entorhinal-hippocampal circuit, including place, head direction, and grid cells, showed 2D activity patterns similar to those in the real world. Furthermore, border cells were observed, and hippocampal remapping was driven by environment shape, suggesting functional processing of virtual boundaries. These results illustrate that 2D spatial representations can be engaged by visual and rotati...
To investigate the mechanisms of persistent neural activity, we obtained in vivo intracellular re... more To investigate the mechanisms of persistent neural activity, we obtained in vivo intracellular recordings from neurons in an oculomotor neural integrator of the goldfish during spontaneous saccades and fixations. Persistent changes in firing rate following saccades were associated with step changes in interspike membrane potential that were correlated with changes in eye position. Perturbation of persistent activity with brief intracellular current pulses designed to mimic saccadic input only induced transient changes of firing rate and membrane potential. When neurons were hyperpolarized below action potential threshold, position-correlated step changes in membrane potential remained. Membrane potential fluctuations were greater during more depolarized steps. These results suggest that sustained changes in firing rate are supported not by either membrane multistability or changes in pacemaker currents, but rather by persistent changes in the rate or amplitude of synaptic inputs.
In layer 2/3 pyramidal neurons of barrel cortex in vivo, calcium ion concentration ([Ca2+]) trans... more In layer 2/3 pyramidal neurons of barrel cortex in vivo, calcium ion concentration ([Ca2+]) transients in apical dendrites evoked by sodium action potentials are limited to regions close to the soma. To study the mechanisms underlying this restricted pattern of calcium influx, we combined two-photon imaging of dendritic [Ca2+] dynamics with dendritic membrane potential measurements. We found that sodium action potentials attenuated and broadened rapidly with distance from the soma. However, dendrites of layer 2/3 cells were electrically excitable, and direct current injections could evoke large [Ca2+] transients. The restricted pattern of dendritic [Ca2+] transients is therefore due to a failure of sodium action-potential propagation into dendrites. Also, stimulating subcortical activating systems by tail pinch can enhance dendritic [Ca2+] influx induced by a sensory stimulus by increasing cellular excitability, consistent with the importance of these systems in plasticity and learn...
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