Imaging GFP-Based Reporters in Neurons with Multiwavelength Optogenetic Control

To study the impact of neural activity on cellular physiology, one would like to combine precise control of firing patterns with highly sensitive probes of cellular physiology. Light-gated ion channels, e.g., Channelrhodopsin-2, enable precise control of firing patterns; green fluorescent protein-based reporters, e.g., the GCaMP6f Ca²⁺ reporter, enable highly sensitive probing of cellular physiology. However, for most actuator-reporter combinations, spectral overlap prevents straightforward combination within a single cell. Here we explore multiwavelength control of channelrhodopsins to circumvent this limitation. The “stoplight” technique described in this article uses channelrhodopsin variants that are opened by blue light and closed by orange light. Cells are illuminated with constant blue light to excite fluorescence of a green fluorescent protein-based reporter. Modulated illumination with orange light negatively regulates activation of the channelrhodopsin. We performed detailed photophysical characterization and kinetic modeling of four candidate stoplight channelrhodopsins. The variant with the highest contrast, sdChR(C138S,E154A), enabled all-optical measurements of activity-induced calcium transients in cultured rat hippocampal neurons, although cell-to-cell variation in expression levels presents a challenge for quantification.     

Imaging GFP-Based Reporters in Neurons with Multiwavelength Optogenetic Control

To study the impact of neural activity on cellular physiology, one would like to combine precise control of firing patterns with highly sensitive probes of cellular physiology. Light-gated ion channels, e.g., Channelrhodopsin-2, enable precise control of firing patterns; green fluorescent protein-based reporters, e.g., the GCaMP6f Ca²⁺ reporter, enable highly sensitive probing of cellular physiology. However, for most actuator-reporter combinations, spectral overlap prevents straightforward combination within a single cell. Here we explore multiwavelength control of channelrhodopsins to circumvent this limitation. The “stoplight” technique described in this article uses channelrhodopsin variants that are opened by blue light and closed by orange light. Cells are illuminated with constant blue light to excite fluorescence of a green fluorescent protein-based reporter. Modulated illumination with orange light negatively regulates activation of the channelrhodopsin. We performed detailed photophysical characterization and kinetic modeling of four candidate stoplight channelrhodopsins. The variant with the highest contrast, sdChR(C138S,E154A), enabled all-optical measurements of activity-induced calcium transients in cultured rat hippocampal neurons, although cell-to-cell variation in expression levels presents a challenge for quantification.     

Aplysia Ganglia Preparation for Electrophysiological and Molecular Analyses of Single Neurons

A major challenge in neurobiology is to understand the molecular underpinnings of neural circuitry that govern a specific behavior. Once the specific molecular mechanisms are identified, new therapeutic strategies can be developed to treat abnormalities in specific behaviors caused by degenerative diseases or aging of the nervous system. The marine snail Aplysia californica is well suited for the investigations of cellular and molecular basis of behavior because neural circuitry underlying a specific behavior could be easily determined and the individual components of the circuitry could be easily manipulated. These advantages of Aplysia have led to several fundamental discoveries of neurobiology of learning and memory. Here we describe a preparation of the Aplysia nervous system for the electrophysiological and molecular analyses of individual neurons. Briefly, ganglion dissected from the nervous system is exposed to protease to remove the ganglion sheath such that neurons are exposed but retain neuronal activity as in the intact animal. This preparation is used to carry out electrophysiological measurements of single or multiple neurons. Importantly, following the recording using a simple methodology, the neurons could be isolated directly from the ganglia for gene expression analysis. These protocols were used to carry out simultaneous electrophysiological recordings from L7 and R15 neurons, study their response to acetylcholine and quantitating expression of CREB1 gene in isolated single L7, L11, R15, and R2 neurons of Aplysia.     

The brain activity map project and the challenge of functional connectomics

The function of neural circuits is an emergent property that arises from the coordinated activity of large numbers of neurons. To capture this, we propose launching a large-scale, international public effort, the Brain Activity Map Project, aimed at reconstructing the full record of neural activity across complete neural circuits. This technological challenge could prove to be an invaluable step toward understanding fundamental and pathological brain processes.     

Cut-loading: A Useful Tool for Examining the Extent of Gap Junction Tracer Coupling Between Retinal Neurons

In addition to chemical synaptic transmission, neurons that are connected by gap junctions can also communicate rapidly via electrical synaptic transmission. Increasing evidence indicates that gap junctions not only permit electrical current flow and synchronous activity between interconnected or coupled cells, but that the strength or effectiveness of electrical communication between coupled cells can be modulated to a great extent^1,2. In addition, the large internal diameter (~1.2 nm) of many gap junction channels permits not only electric current flow, but also the diffusion of intracellular signaling molecules and small metabolites between interconnected cells, so that gap junctions may also mediate metabolic and chemical communication. The strength of gap junctional communication between neurons and its modulation by neurotransmitters and other factors can be studied by simultaneously electrically recording from coupled cells and by determining the extent of diffusion of tracer molecules, which are gap junction permeable, but not membrane permeable, following iontophoretic injection into single cells. However, these procedures can be extremely difficult to perform on neurons with small somata in intact neural tissue.Numerous studies on electrical synapses and the modulation of electrical communication have been conducted in the vertebrate retina, since each of the five retinal neuron types is electrically connected by gap junctions^3,4. Increasing evidence has shown that the circadian (24-hour) clock in the retina and changes in light stimulation regulate gap junction coupling^3-8. For example, recent work has demonstrated that the retinal circadian clock decreases gap junction coupling between rod and cone photoreceptor cells during the day by increasing dopamine D2 receptor activation, and dramatically increases rod-cone coupling at night by reducing D2 receptor activation^7,8. However, not only are these studies extremely difficult to perform on neurons with small somata in intact neural retinal tissue, but it can be difficult to adequately control the illumination conditions during the electrophysiological study of single retinal neurons to avoid light-induced changes in gap junction conductance.Here, we present a straightforward method of determining the extent of gap junction tracer coupling between retinal neurons under different illumination conditions and at different times of the day and night. This cut-loading technique is a modification of scrape loading^9-12, which is based on dye loading and diffusion through open gap junction channels. Scrape loading works well in cultured cells, but not in thick slices such as intact retinas. The cut-loading technique has been used to study photoreceptor coupling in intact fish and mammalian retinas^{7, 8,13}, and can be used to study coupling between other retinal neurons, as described here.     

Escape behavior elicited by single, channelrhodopsin-2-evoked spikes in zebrafish somatosensory neurons

Somatosensory neurons in teleosts and amphibians are sensitive to thermal, mechanical, or nociceptive stimuli [1, 2]. The two main types of such cells in zebrafish--Rohon-Beard and trigeminal neurons--have served as models for neural development [3-6], but little is known about how they encode tactile stimuli. The hindbrain networks that transduce somatosensory stimuli into a motor output encode information by using very few spikes in a small number of cells [7], but it is unclear whether activity in the primary receptor neurons is similarly efficient. To address this question, we manipulated the activity of zebrafish neurons with the light-activated cation channel, Channelrhodopsin-2 (ChR2) [8, 9]. We found that photoactivation of ChR2 in genetically defined populations of somatosensory neurons triggered escape behaviors in 24-hr-old zebrafish. Electrophysiological recordings from ChR2-positive trigeminal neurons in intact fish revealed that these cells have extremely low rates of spontaneous activity and can be induced to fire by brief pulses of blue light. Using this technique, we find that even a single action potential in a single sensory neuron was at times sufficient to evoke an escape behavior. These results establish ChR2 as a powerful tool for the manipulation of neural activity in zebrafish and reveal a degree of efficiency in coding that has not been found in primary sensory neurons.     

Energy coding in neural network with inhibitory neurons

This paper aimed at assessing and comparing the effects of the inhibitory neurons in the neural network on the neural energy distribution, and the network activities in the absence of the inhibitory neurons to understand the nature of neural energy distribution and neural energy coding. Stimulus, synchronous oscillation has significant difference between neural networks with and without inhibitory neurons, and this difference can be quantitatively evaluated by the characteristic energy distribution. In addition, the synchronous oscillation difference of the neural activity can be quantitatively described by change of the energy distribution if the network parameters are gradually adjusted. Compared with traditional method of correlation coefficient analysis, the quantitative indicators based on nervous energy distribution characteristics are more effective in reflecting the dynamic features of the neural network activities. Meanwhile, this neural coding method from a global perspective of neural activity effectively avoids the current defects of neural encoding and decoding theory and enormous difficulties encountered. Our studies have shown that neural energy coding is a new coding theory with high efficiency and great potential.     

Neuromodulation to the Rescue: Compensation of Temperature-Induced Breakdown of Rhythmic Motor Patterns via Extrinsic Neuromodulatory Input

Stable rhythmic neural activity depends on the well-coordinated interplay of synaptic and cell-intrinsic conductances. Since all biophysical processes are temperature dependent, this interplay is challenged during temperature fluctuations. How the nervous system remains functional during temperature perturbations remains mostly unknown. We present a hitherto unknown mechanism of how temperature-induced changes in neural networks are compensated by changing their neuromodulatory state: activation of neuromodulatory pathways establishes a dynamic coregulation of synaptic and intrinsic conductances with opposing effects on neuronal activity when temperature changes, hence rescuing neuronal activity. Using the well-studied gastric mill pattern generator of the crab, we show that modest temperature increase can abolish rhythmic activity in isolated neural circuits due to increased leak currents in rhythm-generating neurons. Dynamic clamp-mediated addition of leak currents was sufficient to stop neuronal oscillations at low temperatures, and subtraction of additional leak currents at elevated temperatures was sufficient to rescue the rhythm. Despite the apparent sensitivity of the isolated nervous system to temperature fluctuations, the rhythm could be stabilized by activating extrinsic neuromodulatory inputs from descending projection neurons, a strategy that we indeed found to be implemented in intact animals. In the isolated nervous system, temperature compensation was achieved by stronger extrinsic neuromodulatory input from projection neurons or by augmenting projection neuron influence via bath application of the peptide cotransmitter Cancer borealis tachykinin-related peptide Ia (CabTRP Ia). CabTRP Ia activates the modulator-induced current I_MI (a nonlinear voltage-gated inward current) that effectively acted as a negative leak current and counterbalanced the temperature-induced leak to rescue neuronal oscillations. Computational modelling revealed the ability of I_MI to reduce detrimental leak-current influences on neuronal networks over a broad conductance range and indicated that leak and I_MI are closely coregulated in the biological system to enable stable motor patterns. In conclusion, these results show that temperature compensation does not need to be implemented within the network itself but can be conditionally provided by extrinsic neuromodulatory input that counterbalances temperature-induced modifications of circuit-intrinsic properties.     

Monitoring activity in neuronal populations with single-cell resolution in a behaving vertebrate

Vertebrate behaviours are produced by activity in populations of neurons, but the techniques typically used to study activity allow only one or very few nerve cells to be monitored at a time. This limitation has prompted the development of methods of imaging activity in the nervous system. The overall goal of these methods is to image neural activity non-invasively in populations of neurons, ideally with high spatial and temporal resolution. We have moved closer to this goal by using confocal calcium imaging to monitor neural activity in the transparent larvae of zebrafish. Neurons were labelled either by backfilling from injections of the calcium indicator (Calcium Green dextran) into muscle or spinal cord of larvae or by injections into blastomeres early in development. The labelled neurons were bright enough at resting calcium levels to allow the identification of individual neurons in the live, intact fish, based upon their dendritic and axonal morphology. The neurons from the live animal could also be reconstructed in three dimensions for morphometric study. Neurons increased their fluorescence during activity produced by direct electrical stimulation and during escape behaviours elicited by an abrupt touch to the head or tail of the fish. The rise in calcium associated with a single action potential could be detected as an increase in fluorescence of at least 7--10%, but neurons typically showed much larger increases during behaviour. Calcium signals in the dendrites, soma and nucleus could be resolved, especially when using the line-scanning mode, which provides 2-ms temporal resolution. The imaging was used to study activity in populations of motoneurons and hindbrain neurons during the escape behaviour fish use to avoid predators. We found a massive activation of the motoneuron pool and a differential activation of populations of hindbrain neurons during escapes. The latter finding confirms predictions that the activity pattern of hindbrain neurons may help to determine the directionality of the escape. This approach should prove useful for studying the activity of populations of neurons throughout the nervous system in both normal and mutant lines of fish. 1998 © Chapman & Hall     

New optical tools for controlling neuronal activity

A major challenge in understanding the relationship between neural activity and development, and ultimately behavior, is to control simultaneously the activity of either many neurons belonging to specific subsets or specific regions within individual neurons. Optimally, such a technique should be capable of both switching nerve cells on and off within milliseconds in a non-invasive manner, and inducing depolarizations or hyperpolarizations for periods lasting from milliseconds to many seconds. Specific ion conductances in subcellular compartments must also be controlled to bypass signaling cascades in order to regulate precisely cellular events such as synaptic transmission. Light-activated G-protein-coupled receptors and ion channels, which can be genetically manipulated and targeted to neuronal circuits, have the greatest potential to fulfill these requirements.     

Improved expression of halorhodopsin for light-induced silencing of neuronal activity

The ability to control and manipulate neuronal activity within an intact mammalian brain is of key importance for mapping functional connectivity and for dissecting the neural circuitry underlying behaviors. We have previously generated transgenic mice that express channelrhodopsin-2 for light-induced activation of neurons and mapping of neural circuits. Here we describe transgenic mice that express halorhodopsin (NpHR), a light-driven chloride pump that can be used to silence neuronal activity via light. Using the Thy-1 promoter to target NpHR expression to neurons, we found that neurons in these mice expressed high levels of NpHR-YFP and that illumination of cortical pyramidal neurons expressing NpHR-YFP led to rapid, reversible photoinhibition of action potential firing in these cells. However, NpHR-YFP expression led to the formation of numerous intracellular blebs, which may disrupt neuronal function. Labeling of various subcellular markers indicated that the blebs arise from retention of NpHR-YFP in the endoplasmic reticulum. By improving the signal peptide sequence and adding an ER export signal to NpHR-YFP, we eliminated the formation of blebs and dramatically increased the membrane expression of NpHR-YFP. Thus, the improved version of NpHR should serve as an excellent tool for neuronal silencing in vitro and in vivo.     

High Accuracy Decoding of Dynamical Motion from a Large Retinal Population

Motion tracking is a challenge the visual system has to solve by reading out the retinal population. It is still unclear how the information from different neurons can be combined together to estimate the position of an object. Here we recorded a large population of ganglion cells in a dense patch of salamander and guinea pig retinas while displaying a bar moving diffusively. We show that the bar’s position can be reconstructed from retinal activity with a precision in the hyperacuity regime using a linear decoder acting on 100+ cells. We then took advantage of this unprecedented precision to explore the spatial structure of the retina’s population code. The classical view would have suggested that the firing rates of the cells form a moving hill of activity tracking the bar’s position. Instead, we found that most ganglion cells in the salamander fired sparsely and idiosyncratically, so that their neural image did not track the bar. Furthermore, ganglion cell activity spanned an area much larger than predicted by their receptive fields, with cells coding for motion far in their surround. As a result, population redundancy was high, and we could find multiple, disjoint subsets of neurons that encoded the trajectory with high precision. This organization allows for diverse collections of ganglion cells to represent high-accuracy motion information in a form easily read out by downstream neural circuits.     

关于耦合神经元活动时的能量原理

最近美国耶鲁大学的神经科学家们用实验数据表明,哺乳动物大脑皮层中神经信号的传递是一个代价昂贵的能量支出过程,而神经信号的编码是与能量代谢紧密地耦合在一起的,但是到目前为止还无法定量给出神经元活动时的能量函数。在这篇文章中,能量原理被用于神经活动和神经信息处理机制的研究,在电生理实验数据的基础上,建立神经元活动的用能量函数表示的运动方程。结果表明用能量函数表达耦合神经元的阈下电活动和动作电位,数值计算结果与用Hodgkin-Huxley方程所描述的动作电位一致。从而有可能依据能量原理从脑信息处理的角度揭示和理解大脑神经网络系统的信息表现规律。     

Effects of PGE2 in guinea pig colonic myenteric ganglia

PGE(2) is a proinflammatory mediator that can influence many cell types. This study was conducted to determine whether PGE(2) alters the electrical activity of distal colonic myenteric neurons, because colitis is typically associated with altered motility and changes in neural signaling may be involved. The electrical properties of intact myenteric neurons were evaluated with intracellular microelectrodes. Acute application of PGE(2) elicited a prolonged depolarization in both AH and S neurons with little effect on input resistance or electrical excitability. PGE(2) effects were suppressed by tetrodotoxin (TTX) or neurokinin (NK) receptor antagonists, indicating that PGE(2) acts directly and indirectly to depolarize colonic neurons. PGE(2)-evoked depolarization was concentration dependent (approximately 3 microM EC(50)) and was attenuated by the E prostanoid (EP)1/2 receptor antagonist, AH-6809. When preparations were maintained for 48 h in the presence of the stable PGE(2) analog PGE(2)-ethanolamide (10 microM), neurons exhibited a significant membrane depolarization and enhanced excitability. These results suggest that PGE(2) can play a role in altered motility in colitis by evoking changes in the electrical properties of myenteric neurons.     

Impact of network structure and cellular response on spike time correlations

Novel experimental techniques reveal the simultaneous activity of larger and larger numbers of neurons. As a result there is increasing interest in the structure of cooperative--or correlated--activity in neural populations, and in the possible impact of such correlations on the neural code. A fundamental theoretical challenge is to understand how the architecture of network connectivity along with the dynamical properties of single cells shape the magnitude and timescale of correlations. We provide a general approach to this problem by extending prior techniques based on linear response theory. We consider networks of general integrate-and-fire cells with arbitrary architecture, and provide explicit expressions for the approximate cross-correlation between constituent cells. These correlations depend strongly on the operating point (input mean and variance) of the neurons, even when connectivity is fixed. Moreover, the approximations admit an expansion in powers of the matrices that describe the network architecture. This expansion can be readily interpreted in terms of paths between different cells. We apply our results to large excitatory-inhibitory networks, and demonstrate first how precise balance--or lack thereof--between the strengths and timescales of excitatory and inhibitory synapses is reflected in the overall correlation structure of the network. We then derive explicit expressions for the average correlation structure in randomly connected networks. These expressions help to identify the important factors that shape coordinated neural activity in such networks.     

Genetic manipulation of the odor-evoked distributed neural activity in the Drosophila mushroom body

Odor-induced neural activity was recorded by Ca2+ imaging in the cell body region of the Drosophila mushroom body (MB), which is the second relay of the olfactory central nervous system. The signals recorded are mainly from the cell layers on the brain surface because of the limited penetration of Ca2+-sensitive dyes. The densely packed cell bodies and their accessibility allow visualization of odor-induced population neural activity. It is revealed that odors evoke diffused neural activities in the MB. Although the signals cannot be attributed to individual neurons, patterns of the population neural activity can be analyzed. The activity pattern, but not the amplitude, of an odor-induced population response is specific for the chemical identity of an odor and its concentration. The distribution pattern of neural activity can be altered specifically by genetic manipulation of an odor binding protein and this alteration is closely associated with a behavioral defect of odor preference. These results suggest that the spatial pattern of the distributed neural activity may contribute to coding of odor information at the second relay of the olfactory system.     

Neural Signatures: Multiple Coding in Spiking–bursting Cells

Recent experiments have revealed the existence of neural signatures in the activity of individual cells of the pyloric central pattern generator (CPG) of crustacean. The neural signatures consist of cell-specific spike timings in the bursting activity of the neurons. The role of these intraburst neural fingerprints is still unclear. It has been reported previously that some muscles can reflect small changes in the spike timings of the neurons that innervate them. However, it is unclear to what extent neural signatures contribute to the command message that the muscles receive from the motoneurons. It is also unknown whether the signatures have any functional meaning for the neurons that belong to the same CPG or to other interconnected CPGs. In this paper, we use realistic neural models to study the ability of single cells and small circuits to recognize individual neural signatures. We show that model cells and circuits can respond distinctly to the incoming neural fingerprints in addition to the properties of the slow depolarizing waves. Our results suggest that neural signatures can be a general mechanism of spiking–bursting cells to implement multicoding. An erratum to this article can be found at     

Optogenetics and thermogenetics: technologies for controlling the activity of targeted cells within intact neural circuits

In recent years, interest has grown in the ability to manipulate, in a temporally precise fashion, the electrical activity of specific neurons embedded within densely wired brain circuits, in order to reveal how specific neurons subserve behaviors and neural computations, and to open up new horizons on the clinical treatment of brain disorders. Technologies that enable temporally precise control of electrical activity of specific neurons, and not these neurons' neighbors-whose cell bodies or processes might be just tens to hundreds of nanometers away-must involve two components. First, they require as a trigger a transient pulse of energy that supports the temporal precision of the control. Second, they require a molecular sensitizer that can be expressed in specific neurons and which renders those neurons specifically responsive to the triggering energy delivered. Optogenetic tools, such as microbial opsins, can be used to activate or silence neural activity with brief pulses of light. Thermogenetic tools, such as thermosensitive TRP channels, can be used to drive neural activity downstream of increases or decreases in temperature. We here discuss the principles underlying the operation of these two recently developed, but widely used, toolboxes, as well as the directions being taken in the use and improvement of these toolboxes.     

Neural network features distinguish chemosensory stimuli in Caenorhabditis elegans

Nervous systems extract and process information from the environment to alter animal behavior and physiology. Despite progress in understanding how different stimuli are represented by changes in neuronal activity, less is known about how they affect broader neural network properties. We developed a framework for using graph-theoretic features of neural network activity to predict ecologically relevant stimulus properties, in particular stimulus identity. We used the transparent nematode, Caenorhabditis elegans, with its small nervous system to define neural network features associated with various chemosensory stimuli. We first immobilized animals using a microfluidic device and exposed their noses to chemical stimuli while monitoring changes in neural activity of more than 50 neurons in the head region. We found that graph-theoretic features, which capture patterns of interactions between neurons, are modulated by stimulus identity. Further, we show that a simple machine learning classifier trained using graph-theoretic features alone, or in combination with neural activity features, can accurately predict salt stimulus. Moreover, by focusing on putative causal interactions between neurons, the graph-theoretic features were almost twice as predictive as the neural activity features. These results reveal that stimulus identity modulates the broad, network-level organization of the nervous system, and that graph theory can be used to characterize these changes.     

Central regulation of bone mass

The traditional view of skeletal homeostasis as a primarily endocrine activity has been expanded in recent years following the identification of direct neural pathways controlling bone homeostasis via central relays. Powerful control over both anabolic and catabolic activities have been isolated to neurons of the hypothalamus, enabling large changes in bone mass to be achieved by minute changes in the levels of these central neural signals. Initiated by studies of leptin and expanding rapidly, the breadth and complexity of this regulatory axis to bone is sure to increase. Critically though, the translation of these findings into therapeutic interventions is likely to present a greater challenge. However, the contribution to our understanding that these initial studies are making indicates an exciting potential to help to alleviate the growing challenge presented by musculoskeletal disease.