Visualizing Protein Kinase A Activity In Head-fixed Behaving Mice Using In Vivo Two-photon Fluorescence Lifetime Imaging Microscopy
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Neuromodulation exerts powerful control over brain function. Dysfunction of neuromodulatory systems results in neurological and psychiatric disorders. Despite their importance, technologies for tracking neuromodulatory events with cellular resolution are just beginning to emerge. Neuromodulators, such as dopamine, norepinephrine, acetylcholine, and serotonin, trigger intracellular signaling events via their respective G protein-coupled receptors to modulate neuronal excitability, synaptic communications, and other neuronal functions, thereby regulating information processing in the neuronal network. The above mentioned neuromodulators converge onto the cAMP/protein kinase A (PKA) pathway. Therefore, in vivo PKA imaging with single-cell resolution was developed as a readout for neuromodulatory events in a manner analogous to calcium imaging for neuronal electrical activities. Herein, a method is presented to visualize PKA activity at the level of individual neurons in the cortex of head-fixed behaving mice. To do so, an improved A-kinase activity reporter (AKAR), called tAKARα, is used, which is based on Förster resonance energy transfer (FRET). This genetically-encoded PKA sensor is introduced into the motor cortex via in utero electroporation (IUE) of DNA plasmids, or stereotaxic injection of adeno-associated virus (AAV). FRET changes are imaged using two-photon fluorescence lifetime imaging microscopy (2pFLIM), which offers advantages over ratiometric FRET measurements for quantifying FRET signal in light-scattering brain tissue. To study PKA activities during enforced locomotion, tAKARα is imaged through a chronic cranial window above the cortex of awake, head-fixed mice, which run or rest on a speed-controlled motorized treadmill. This imaging approach will be applicable to many other brain regions to study corresponding behavior-induced PKA activities and to other FLIM-based sensors for in vivo imaging.Keywords:
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The mammalian brain is reported to contain about 10 6 –10 9 neurons linked together to form complex networks. Physiologically, the neuronal networks interact in a rhythmic oscillatory pattern to coordinate the brain’s functions. Neuromodulation covers a broad range of techniques that can alter neuronal network activity through the targeted delivery of electrical or chemical stimuli. Neuromodulation can be used to potentially treat medical conditions and can serve as a research tool for studying neural functions. Typically, the main method of neuromodulation is to electrically stimulate specific structures in both the central and peripheral nervous systems via surgically implanted electrodes. Therefore, it is imperative to explore novel and safer methods for altering neuronal network activity. Transcorneal electrical stimulation (TES) has rapidly emerged as a non-invasive neuromodulatory technique that can exert beneficial effects on the brain through the eyes. There is substantial evidence to show that TES can change the brain oscillations in rodents. Moreover, the molecular data clearly shows that TES can also activate non-visual brain regions. In this review, we first summarize the use of TES in the retina and then discuss its effects in the brain through the eye-brain connection. We then comprehensively review the substantial evidence from electrophysiological, behavioral, and molecular studies on the role of TES on modulating neurons in the brain. Lastly, we discuss the implications and possible future directions of the research on TES as a non-invasive tool for neuromodulation of the brain via directly stimulating the mammalian eye.
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Abstract Astrocytes contact thousands of synapses throughout the territory covered by its fine bushy processes. Astrocytes respond to neuronal activity with an increase in calcium concentration that is in turn linked to their capacity to modulate neuronal activity. It remains unclear whether astrocytes behave as a single functional unit that integrates all of these inputs, or if multiple functional subdomains reside within an individual astrocyte. We utilized the topographic organization of ferret visual cortex to test whether local neuronal activity can elicit spatially restricted events within an individual astrocyte. We monitored calcium activity throughout the extent of astrocytes in ferret visual cortex while presenting visual stimuli that elicit coordinated neuronal activity spatially restricted to functional columns. We found visually-driven calcium responses throughout the entire astrocyte that was largely independent in individual subdomains, often responding to different visual stimulus orientations. A model of the spatial interaction of astrocytes and neuronal orientation maps recapitulated these measurements, consistent with the hypothesis that astrocyte subdomains integrate local neuronal activity. Together, these results suggest that astrocyte responses to neural circuit activity are dominated by functional subdomains that respond locally and independently to neuronal activity.
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Neuromodulators play an important role in how the nervous system organizes activity that results in behavior. Disruption of the normal patterns of neuromodulatory release or production is known to be related to the onset of severe pathologies such as Parkinson’s disease, Rett syndrome, Alzheimer’s disease, and affective disorders. Some of these pathologies involve neuronal structures that are called central pattern generators (CPGs), which are involved in the production of rhythmic activities throughout the nervous system. Here I discuss the interplay between CPGs and neuromodulatory activity, with particular emphasis on the potential role of neuromodulators in the recovery of disrupted neuronal activity. I refer to invertebrate and vertebrate model systems and some of the lessons we have learned from research on these systems and propose a few avenues for future research. I make one suggestion that may guide future research in the field: neuromodulators restrict the parameter landscape in which CPG components operate, and the removal of neuromodulators may enable a perturbed CPG in finding a new set of parameter values that can allow it to regain normal function.
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Abstract Electrical stimulation of the brain has become a mainstay of fundamental neuroscience research and an increasingly prevalent clinical therapy. Despite decades of use in basic neuroscience research and the growing prevalence of neuromodulation therapies, gaps in knowledge regarding activation or inactivation of neural elements over time have limited its ability to adequately interpret evoked downstream responses or fine-tune stimulation parameters to focus on desired responses. In this work, in vivo two-photon microscopy was used to image neuronal calcium activity in layer 2/3 neurons of somatosensory cortex (S1) in male C57BL/6J-Tg(Thy1-GCaMP6s)GP4.3Dkim/J mice during 30 s of continuous electrical stimulation at varying frequencies. We show frequency-dependent differences in spatial and temporal somatic responses during continuous stimulation. Our results elucidate conflicting results from prior studies reporting either dense spherical activation of somas biased towards those near the electrode, or sparse activation of somas at a distance via axons near the electrode. These findings indicate that the neural element specific temporal response local to the stimulating electrode changes as a function of applied charge density and frequency. These temporal responses need to be considered to properly interpret downstream circuit responses or determining mechanisms of action in basic science experiments or clinical therapeutic applications. Significance Statement Microstimulation of small populations of neurons has the potential to ameliorate symptoms associated with several neurological disorders. However, the specific mechanisms by which microstimulation elicits therapeutic responses are unclear. This work examines the effects of continuous microstimulation on the local population of neurons surrounding the implanted electrode. Stimulation was found to elicit spatiotemporal neuronal responses in a frequency dependent manner. These findings suggest that stimulation frequency may be an important consideration for applications in research or therapy. Further research aimed at understanding these neuronal activation properties may provide insight into the mechanistic mode of action of continuous microstimulation.
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The first three postnatal weeks in rodents are a time when sensory experience drives the maturation of brain circuits, an important process that is not yet well understood. Alterations in this critical period of experience-dependent circuit assembly and plasticity contribute to several neurodevelopmental disorders, such as autism, epilepsy, and schizophrenia. Therefore, techniques for recording network activity and tracing neuronal connectivity over this time period are necessary for delineating circuit refinement in typical development and how it deviates in disease. Calcium imaging with GCaMP6 and other genetically encoded indicators is rapidly becoming the preferred method for recording network activity at the single-synapse and single-cell level in vivo, especially in genetically identified neuronal populations. We describe a protocol for intracortical injection of recombinant adeno-associated viruses in P1 neonatal mice and demonstrate its use for longitudinal imaging of GCaMP6s in the same neurons over several weeks to characterize the developmental desynchronization of cortical network activity. Our approach is ideally suited for chronic in vivo two-photon calcium imaging of neuronal activity from synapses to entire networks during the early postnatal period.
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There are many sources of modulatory input to CPGs and other types of neuronal circuits. These inputs can change the properties of cells and synapses and dramatically alter the production of motor patterns. Sometimes this enables the production of motor patterns by the circuit. At other times, the modulation allows alternate motor patterns to be produced by a single circuit. Modulatory neurones have fast as well as slow actions. In some cases, such as with GPR, the two types of effects are due to the release of co‐transmitters. In other cases, such as with the DSIs, a single substance can act at different receptors to cause fast and slow postsynaptic actions. The effect of a neuromodulatory neurone is determined by the type of receptor on the target neurone. Thus a single modulatory neurone evokes a suite of actions in a circuit and thereby produces a co‐ordinated output. Extrinsic and intrinsic sources of neuromodulation have different sets of constraints acting upon them. For example, extrinsic neuromodulation can easily be used for motor pattern selection; a different pattern is produced depending upon which modulatory inputs are active. However, intrinsic neuromodulation is not well suited to that task. Instead, it is useful for self‐organizing properties and experience‐dependent effects. One clear conclusion from this work and other work in the field is that neuromodulation by neurones intrinsic and extrinsic to CPGs is not uncommon (Katz, 1995; Katz & Frost, 1996). It is part of the normal process of motor pattern generation. As such, it needs to be considered when discussing mechanisms for neuronal circuit actions.
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The neural code that relates the firing of neurons to the generation of behavior and mental states must be implemented by spatiotemporal patterns of activity across neuronal populations. These patterns engage selective groups of neurons, called neuronal ensembles, which are emergent building blocks of neural circuits. We review optical and computational methods, based on two-photon calcium imaging and two-photon optogenetics, to detect, characterize, and manipulate neuronal ensembles in three dimensions. We review data using these methods in the mammalian cortex that demonstrate the existence of neuronal ensembles in the spontaneous and evoked cortical activity in vitro and in vivo. Moreover, two-photon optogenetics enable the possibility of artificially imprinting neuronal ensembles into awake, behaving animals and of later recalling those ensembles selectively by stimulating individual cells. These methods could enable deciphering the neural code and also be used to understand the pathophysiology of and design novel therapies for neurological and mental diseases.
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Many nervous system disorders (e.g., Parkinson's disease, mood disorders) involve neurotransmitters as well as electrical activity. Pharmacologic treatment does not target the precise location(s) where neurotransmitter imbalances occur. Additionally, non-neuronal cells in the brain--notably astrocytes--influence neuronal activity through both electrical and neurochemical modulation of nearby neurons. Precise monitoring/recording and modulating/stimulating (both electrical and neurochemical) can optimize therapy in specific disorders and specific patients. Carbon-fiber microelectrodes (5 microm diameter) in freely moving rodents have shown that dopamine release is heterogeneous within various regions in the nucleus accumbens, a region involved in many mood disorders. Because neurons are only several microns in diameter (axons, dendrites, and synaptic clefts smaller still), ultramicroelectrodes will be essential to selectively monitor/modulate the cell body, the axon, or at the intracellular level. Nanoelectrode arrays can monitor both electrical activity and dopamine in real time with submicron resolution, and stimulate neurons with equal precision. Computational models indicate that precise monitoring/modulating (electrically and neurochemically) at the subnucleus or neuron level will be necessary to restore normal firing patterns and neurotransmitter levels in many brain disorders. Endovascular techniques can introduce ultramicroelectrodes (0.5 micron or smaller) into the brain via capillaries; such electrodes can stimulate/record neuronal tissue with a response virtually identical to extra-vascular microelectrodes. Within the next decade, hundreds if not thousands of submicron-sized monitoring/modulating electrodes can be placed wherever needed to restore brain function to normal. The term "neuromodulation" will likely replace deep brain stimulation (DBS) as both neurochemistry and electrical activity are included in the therapeutic modalities.
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