Two-photon Ca2 Research Articles

Comparing confocal and two-photon Ca2+ imaging of thin low-scattering preparations

Ca2+ imaging provides insight into biological processes ranging from subcellular dynamics to neural network activity. Two-photon microscopy has assumed a dominant role in Ca2+ imaging. The longer wavelength infra-red illumination undergoes less scattering, and absorption is confined to the focal plane. Two-photon imaging can thus penetrate thick tissue ∼10-fold more deeply than single-photon visible imaging to make two-photon microscopy an exceptionally powerful method for probing function in intact brain. However, two-photon excitation produces photobleaching and photodamage that increase very steeply with light intensity, limiting how strongly one can illuminate. In thin samples, illumination intensity can assume a dominant role in determining signal quality, raising the possibility that single-photon microscopy may be preferable. We therefore tested laser scanning single-photon and two-photon microscopy side by side with Ca2+ imaging in neuronal compartments at the surface of a brain slice. We optimized illumination intensity for each light source to obtain the brightest signal without photobleaching. Intracellular Ca2+ rises elicited by one action potential had twice the signal/noise ratio with confocal as with two-photon imaging in axons, were 31% higher in dendrites, and about the same in cell bodies. The superior performance of confocal imaging in finer neuronal processes likely reflects the dominance of shot noise when fluorescence is dim. Thus, when out-of-focus absorption and scattering are not issues, single-photon confocal imaging can yield better quality signals than two-photon microscopy.

NeuroSeg-II: A deep learning approach for generalized neuron segmentation in two-photon Ca2+ imaging.

The development of two-photon microscopy and Ca2+ indicators has enabled the recording of multiscale neuronal activities in vivo and thus advanced the understanding of brain functions. However, it is challenging to perform automatic, accurate, and generalized neuron segmentation when processing a large amount of imaging data. Here, we propose a novel deep-learning-based neural network, termed as NeuroSeg-II, to conduct automatic neuron segmentation for in vivo two-photon Ca2+ imaging data. This network architecture is based on Mask region-based convolutional neural network (R-CNN) but has enhancements of an attention mechanism and modified feature hierarchy modules. We added an attention mechanism module to focus the computation on neuron regions in imaging data. We also enhanced the feature hierarchy to extract feature information at diverse levels. To incorporate both spatial and temporal information in our data processing, we fused the images from average projection and correlation map extracting the temporal information of active neurons, and the integrated information was expressed as two-dimensional (2D) images. To achieve a generalized neuron segmentation, we conducted a hybrid learning strategy by training our model with imaging data from different labs, including multiscale data with different Ca2+ indicators. The results showed that our approach achieved promising segmentation performance across different imaging scales and Ca2+ indicators, even including the challenging data of large field-of-view mesoscopic images. By comparing state-of-the-art neuron segmentation methods for two-photon Ca2+ imaging data, we showed that our approach achieved the highest accuracy with a publicly available dataset. Thus, NeuroSeg-II enables good segmentation accuracy and a convenient training and testing process.

Cocaine induces locomotor sensitization through a dopamine-dependent VTA-mPFC-FrA cortico-cortical pathway in male mice

As a central part of the mammalian brain, the prefrontal cortex (PFC) has been implicated in regulating cocaine-induced behaviors including compulsive seeking and reinstatement. Although dysfunction of the PFC has been reported in animal and human users with chronic cocaine abuse, less is known about how the PFC is involved in cocaine-induced behaviors. By using two-photon Ca2+ imaging to simultaneously record tens of intact individual networking neurons in the frontal association cortex (FrA) in awake male mice, here we report that a systematic acute cocaine exposure decreased the FrA neural activity in mice, while the chemogenetic intervention blocked the cocaine-induced locomotor sensitization. The hypoactivity of FrA neurons was critically dependent on both dopamine transporters and dopamine transmission in the ventromedial PFC (vmPFC). Both dopamine D1R and D2R neurons in the vmPFC projected to and innervated FrA neurons, the manipulation of which changed the cocaine-induced hypoactivity of the FrA and locomotor sensitization. Together, this work demonstrates acute cocaine-induced hypoactivity of FrA neurons in awake mice, which defines a cortico-cortical projection bridging dopamine transmission and cocaine sensitization.

Daily two-photon neuronal population imaging with targeted single-cell electrophysiology and subcellular imaging in auditory cortex of behaving mice.

Quantitative and mechanistic understanding of learning and long-term memory at the level of single neurons in living brains require highly demanding techniques. A specific need is to precisely label one cell whose firing output property is pinpointed amidst a functionally characterized large population of neurons through the learning process and then investigate the distribution and properties of dendritic inputs. Here, we disseminate an integrated method of daily two-photon neuronal population Ca2+ imaging through an auditory associative learning course, followed by targeted single-cell loose-patch recording and electroporation of plasmid for enhanced chronic Ca2+ imaging of dendritic spines in the targeted cell. Our method provides a unique solution to the demand, opening a solid path toward the hard-cores of how learning and long-term memory are physiologically carried out at the level of single neurons and synapses.

Experience-dependent functional plasticity and visual response selectivity of surviving subplate neurons in the mouse visual cortex

Subplate neurons are early-born cortical neurons that transiently form neural circuits during perinatal development and guide cortical maturation. Thereafter, most subplate neurons undergo cell death, while some survive and renew their target areas for synaptic connections. However, the functional properties of the surviving subplate neurons remain largely unknown. This study aimed to characterize the visual responses and experience-dependent functional plasticity of layer 6b (L6b) neurons, the remnants of subplate neurons, in the primary visual cortex (V1). Two-photon Ca2+ imaging was performed in V1 of awake juvenile mice. L6b neurons showed broader tunings for orientation, direction, and spatial frequency than did layer 2/3 (L2/3) and L6a neurons. In addition, L6b neurons showed lower matching of preferred orientation between the left and right eyes compared with other layers. Post hoc 3D immunohistochemistry confirmed that the majority of recorded L6b neurons expressed connective tissue growth factor (CTGF), a subplate neuron marker. Moreover, chronic two-photon imaging showed that L6b neurons exhibited ocular dominance (OD) plasticity by monocular deprivation during critical periods. The OD shift to the open eye depended on the response strength to the stimulation of the eye to be deprived before starting monocular deprivation. There were no significant differences in visual response selectivity prior to monocular deprivation between the OD changed and unchanged neuron groups, suggesting that OD plasticity can occur in L6b neurons showing any response features. In conclusion, our results provide strong evidence that surviving subplate neurons exhibit sensory responses and experience-dependent plasticity at a relatively late stage of cortical development.

Altered brain state during episodic dystonia in tottering mice decouples primary motor cortex from limb kinematics.

Episodic Ataxia Type 2 (EA2) is a rare neurological disorder caused by a mutation in the CACNA1A gene, encoding the P/Q-type voltage-gated Ca2+ channel important for neurotransmitter release. Patients with this channelopathy exhibit both cerebellar and cerebral pathologies, suggesting the condition affects both regions. The tottering (tg/tg) mouse is the most commonly used EA2 model due to an orthologous mutation in the cacna1a gene. The tg/tg mouse has three prominent behavioral phenotypes: a dramatic episodic dystonia; absence seizures with generalized spike and wave discharges (GSWDs); and mild ataxia. We previously observed a novel brain state, transient low-frequency oscillations (LFOs) in the cerebellum and cerebral cortex under anesthesia. In this study, we examine the relationships among the dystonic attack, GSWDs, and LFOs in the cerebral cortex. Previous studies characterized LFOs in the motor cortex of anesthetized tg/tg mice using flavoprotein autofluorescence imaging testing the hypothesis that LFOs provide a mechanism for the paroxysmal dystonia. We sought to obtain a more direct understanding of motor cortex (M1) activity during the dystonic episodes. Using two-photon Ca2+ imaging to investigate neuronal activity in M1 before, during, and after the dystonic attack, we show that there is not a significant change in the activity of M1 neurons from baseline through the attack. We also conducted simultaneous, multi-electrode recordings to further understand how M1 cellular activity and local field potentials change throughout the progression of the dystonic attack. Neither putative pyramidal nor inhibitory interneuron firing rate changed during the dystonic attack. However, we did observe a near complete loss of GSWDs during the dystonic attack in M1. Finally, using spike triggered averaging to align simultaneously recorded limb kinematics to the peak Ca2+ response, and vice versa, revealed a reduction in the spike triggered average during the dystonic episodes. Both the loss of GSWDs and the reduction in the coupling suggest that, during the dystonic attack, M1 is effectively decoupled from other structures. Overall, these results indicate that the attack is not initiated or controlled in M1, but elsewhere in the motor circuitry. The findings also highlight that LFOs, GSWDs, and dystonic attacks represent three brain states in tg/tg mice.

Cocaine regulates sensory filtering in cortical pyramidal neurons.

Exposure to cocaine leads to robust changes in the structure and function of neurons within the mesocorticolimbic pathway. However, little is known about how cocaine influences the processing of information within the sensory cortex. We address this by using patch-clamp and juxtacellular voltage recordings and two-photon Ca2+ imaging invivo to investigate the influence of acute cocaine exposure on layer 2/3 (L2/3) pyramidal neurons within the primary somatosensory cortex (S1). Here, cocaine dampens membrane potential state transitions and decreases spontaneous somatic action potentials and Ca2+ transients. In contrast to the uniform decrease in background spontaneous activity, cocaine has a heterogeneous influence on sensory encoding, increasing tactile-evoked responses in dendrites that do not typically encode sensory information and decreasing responses in those dendrites that are more reliable sensory encoders. Combined, these findings suggest that cocaine acts as a filter that suppresses background noise to selectively modulate incoming sensory information.

Lithium Provides Broad Therapeutic Benefits in an Alzheimer's Disease Mouse Model.

Alzheimer's disease (AD) is a chronic neurodegenerative disorder with a progressive loss of cognitive function. Currently, no effective treatment regimen is available. Lithium, a mood stabilizer for bipolar disorder, exerts broad neuroprotective and neurotrophic actions and improves cognitive function. The study investigated if lithium stabilizes Ca2+ signaling abnormalities in hippocampal neurons and subsequently normalize downstream effects on AD neuropathology and synaptic plasticity in young AD mice. Four-month-old 3xTg-AD mice were treated with a LiCl diet chow for 30 days. At the end of the lithium treatment, a combination of two-photon Ca2+ imaging, electrophysiology, and immunohistochemistry assays were used to assess the effects of the LiCl treatment on inositol trisphosphate receptor (IP3R)-dependent endoplasmic reticulum (ER) Ca2+ and voltage-gated Ca2+ channel (VGCC)-mediated Ca2+ signaling in CA1 neurons, neuronal nitric oxide synthase (nNOS) and hyperphosphorylated tau (p-tau) levels and synaptic plasticity in the hippocampus and overlying cortex from 3xTg-ADmice. Thirty-day LiCl treatment reduced aberrant IP3R-dependent ER Ca2+ and VGCC-mediated Ca2+ signaling in CA1 pyramidal neurons from 3xTg-AD mice and restored neuronal nitric oxide synthase (nNOS) and hyperphosphorylated tau (p-tau) levels to control levels in the hippocampal subfields and overlying cortex. The LiCl treatment enhanced post-tetanic potentiation (PTP), a form of short-term plasticity in the hippocampus. The study found that lithium exerts therapeutic effects across several AD-associated early neuronal signaling abnormalities including aberrant Ca2+ signaling, nNOS, and p-tau formation and enhances short-term synaptic plasticity. Lithium could serve as an effective treatment or co-therapeutic for AD.

Network instability dynamics drive a transient bursting period in the developing hippocampus in vivo.

Spontaneous correlated activity is a universal hallmark of immature neural circuits. However, the cellular dynamics and intrinsic mechanisms underlying network burstiness in the intact developing brain are largely unknown. Here, we use two-photon Ca2+ imaging to comprehensively map the developmental trajectories of spontaneous network activity in the hippocampal area CA1 of mice in vivo. We unexpectedly find that network burstiness peaks after the developmental emergence of effective synaptic inhibition in the second postnatal week. We demonstrate that the enhanced network burstiness reflects an increased functional coupling of individual neurons to local population activity. However, pairwise neuronal correlations are low, and network bursts (NBs) recruit CA1 pyramidal cells in a virtually random manner. Using a dynamic systems modeling approach, we reconcile these experimental findings and identify network bi-stability as a potential regime underlying network burstiness at this age. Our analyses reveal an important role of synaptic input characteristics and network instability dynamics for NB generation. Collectively, our data suggest a mechanism, whereby developing CA1 performs extensive input-discrimination learning prior to the onset of environmental exploration.

Methcathinone Increases Visually-evoked Neuronal Activity and Enhances Sensory Processing Efficiency in Mice.

Methcathinone (MCAT) belongs to the designer drugs called synthetic cathinones, which are abused worldwide for recreational purposes. It has strong stimulant effects, including enhanced euphoria, sensation, alertness, and empathy. However, little is known about how MCAT modulates neuronal activity in vivo. Here, we evaluated the effect of MCAT on neuronal activity with a series of functional approaches. C-Fos immunostaining showed that MCAT increased the number of activated neurons by 6-fold, especially in sensory and motor cortices, striatum, and midbrain motor nuclei. In vivo single-unit recording and two-photon Ca2+ imaging revealed that a large proportion of neurons increased spiking activity upon MCAT administration. Notably, MCAT induced a strong de-correlation of population activity and increased trial-to-trial reliability, specifically during a natural movie stimulus. It improved the information-processing efficiency by enhancing the single-neuron coding capacity, suggesting a cortical network mechanism of the enhanced perception produced by psychoactive stimulants.

Altered population activity and local tuning heterogeneity in auditory cortex of Cacna2d3-deficient mice.

The α2δ3 auxiliary subunit of voltage-activated calcium channels is required for normal synaptic transmission and precise temporal processing of sounds in theauditory brainstem. In mice its loss additionally leads to an inability to distinguish amplitude-modulated tones. Furthermore, loss of function of α2δ3 has been associated with autism spectrum disorder in humans. To investigate possible alterations of network activity in the higher-order auditory system in α2δ3 knockout mice, we analyzed neuronal activity patterns and topography of frequency tuning within networks of the auditory cortex (AC) using two-photon Ca2+ imaging. Compared to wild-type mice we found distinct subfield-specific alterations in the primary auditory cortex, expressed in overall lower correlations between the network activity patterns in response to different sounds as well as lower reliability of these patterns upon repetitions of the same sound. Higher AC subfields did not display these alterations but showed a higher amount of well-tuned neurons along with lower local heterogeneity of the neurons' frequency tuning. Our results provide new insight into AC network activity alterations in an autism spectrum disorder-associated mouse model.

Astrocytes exhibit diverse Ca2+ changes at subcellular domains during brain aging.

Astrocytic Ca2+ transients are essential for astrocyte integration into neural circuits. These Ca2+ transients are primarily sequestered in subcellular domains, including primary branches, branchlets and leaflets, and endfeet. In previous studies, it suggests that aging causes functional defects in astrocytes. Until now, it was unclear whether and how aging affects astrocytic Ca2+ transients at subcellular domains. In this study, we combined a genetically encoded Ca2+ sensor (GCaMP6f) and in vivo two-photon Ca2+ imaging to determine changes in Ca2+ transients within astrocytic subcellular domains during brain aging. We showed that aging increased Ca2+ transients in astrocytic primary branches, higher-order branchlets, and terminal leaflets. However, Ca2+ transients decreased within astrocytic endfeet during brain aging, which could be caused by the decreased expressions of Aquaporin-4 (AQP4). In addition, aging-induced changes of Ca2+ transient types were heterogeneous within astrocytic subcellular domains. These results demonstrate that the astrocytic Ca2+ transients within subcellular domains are affected by aging differently. This finding contributes to a better understanding of the physiological role of astrocytes in aging-induced neural circuit degeneration.

Evaluation and Manipulation of Neural Activity using Two-Photon Holographic Microscopy.

Recent advances in optical bioimaging and optogenetics have enabled the visualization and manipulation of biological phenomena, including cellular activities, in living animals. In the field of neuroscience, detailed neural activity related to brain functions, such as learning and memory, has now been revealed, and it has become feasible to artificially manipulate this activity to express brain functions. However, the conventional evaluation of neural activity by two-photon Ca2+ imaging has the problem of low temporal resolution. In addition, manipulation of neural activity by conventional optogenetics through the optic fiber can only simultaneously regulate the activity of neurons with the same genetic background, making it difficult to control the activity of individual neurons. To solve this issue, we recently developed a microscope with a high spatiotemporal resolution for biological applications by combining optogenetics with digital holographic technology that can modify femtosecond infrared laser beams. Here, we describe protocols for the visualization, evaluation, and manipulation of neural activity, including the preparation of samples and operation of a two-photon holographic microscope (Figure 1). These protocols provide accurate spatiotemporal information on neural activity, which may be useful for elucidating the pathogenesis of neuropsychiatric disorders that lead to abnormalities in neural activity.

A Standardized Nonvisual Behavioral Event Is Broadcasted Homogeneously across Cortical Visual Areas without Modulating Visual Responses.

Multiple recent studies have shown that motor activity greatly impacts the activity of primary sensory areas like V1. Yet, the role of this motor related activity in sensory processing is still unclear. Here, we dissect how these behavior signals are broadcast to different layers and areas of the visual cortex. To do so, we leveraged a standardized and spontaneous behavioral fidget event in passively viewing mice. Importantly, this behavior event had no relevance to any ongoing task allowing us to compare its neuronal correlates with visually relevant behaviors (e.g., running). A large two-photon Ca2+ imaging database of neuronal responses uncovered four neural response types during fidgets that were consistent in their proportion and response patterns across all visual areas and layers of the visual cortex. Indeed, the layer and area identity could not be decoded above chance level based only on neuronal recordings. In contrast to running behavior, fidget evoked neural responses that were independent to visual processing. The broad availability of visually orthogonal standardized behavior signals could be a key component in how the cortex selects, learns and binds local sensory information with motor outputs. Contrary to behaviorally relevant motor outputs, irrelevant motor signals could project to separate local neural subspaces.

Motor learning-induced new dendritic spines are preferentially involved in the learned task than existing spines.

Learning induces the formation of new synapses in addition to changes of existing synapse strength. However, it remains unclear whether new synapses serve different functions from existing synapses. By performing two-photon structural and Ca2+ imaging of postsynaptic dendritic spines in layer 2/3 pyramidal neurons, we show that new spine formation increases in the mouse motor cortex 8-24h after motor training. New spines, not existing spine populations, are preferentially active when mice perform the learned task rather than a new task. New spine activity is also more synchronized with dendritic/somatic activity when the learned task, not a new task, is carried out. Furthermore, new spines are formed to increase the task specificity in a subset of neurons, and their survival is not affected when a new task is learned. These findings suggest that newly formed synapses preferentially increase the task specificity of neurons over existing synapses at the retention stage of motor learning.

Cell-type-specific silence in thalamocortical circuits precedes hippocampal sharp-wave ripples.

Evidence suggests that the hippocampus conveys memory-related neural patterns across distributed cortical circuits during high-frequency oscillations called sharp-wave ripples (SWRs). We investigate how circuit activity in the retrosplenial cortex (RSC), a primary hippocampal target, could aid in processing SWR-related input. Using patch-clamp recordings from awake mice, we find that SWR-aligned membrane potential modulation is widespread but weak, and that spiking responses are sparse. However, using cell-type-specific two-photon Ca2+ imaging and optogenetics, we show that, 1-2s before SWRs, superficial inhibition and thalamocortical input in RSC is reduced. We propose that pyramidal dendrites experience decreased local inhibition and subcortical interference in a seconds-long time window preceding SWRs. This may aid communication of weak and sparse SWR-aligned excitation between the hippocampus and neocortex and promote the strengthening of memory-related connections.

Using two-photon Ca2+ imaging to reveal spinal cord dorsal horn networks

The spinal cord dorsal horn is a key area for the processing of sensory information such as pain, heat, cold, touch and itch. Our understanding of spinal cord microcircuitry has proved difficult, in part, due to a lack of effective tools to allow identification of dorsal horn neuron populations and how they are connected. A recent technique developed in our lab using pharmacology-based profiling (termed CICADA) has allowed us to visualize eight populations of excitatory neurons in the superficial dorsal horn. Here, we use two-photon Ca2+ imaging and CICADA to tease apart connectivity across these CICADA-defined populations. Through back labeling spinoparabrachial projection neurons, we also identify spinal output neurons, and the spinal networks that provide input onto these cells. These experiments reveal the network connectivity, including the spinal output neurons, that are downstream of each CICADA-defined population. Together, these findings provide new insight into the connectivity in the superficial dorsal horn. Grant support from 2R01 NS096705-06.

Fast and Accurate Motion Correction for Two-Photon Ca2+ Imaging in Behaving Mice.

Two-photon Ca2+ imaging is a widely used technique for investigating brain functions across multiple spatial scales. However, the recording of neuronal activities is affected by movement of the brain during tasks in which the animal is behaving normally. Although post-hoc image registration is the commonly used approach, the recent developments of online neuroscience experiments require real-time image processing with efficient motion correction performance, posing new challenges in neuroinformatics. We propose a fast and accurate image density feature-based motion correction method to address the problem of imaging animal during behaviors. This method is implemented by first robustly estimating and clustering the density features from two-photon images. Then, it takes advantage of the temporal correlation in imaging data to update features of consecutive imaging frames with efficient calculations. Thus, motion artifacts can be quickly and accurately corrected by matching the features and obtaining the transformation parameters for the raw images. Based on this efficient motion correction strategy, our algorithm yields promising computational efficiency on imaging datasets with scales ranging from dendritic spines to neuronal populations. Furthermore, we show that the proposed motion correction method outperforms other methods by evaluating not only computational speed but also the quality of the correction performance. Specifically, we provide a powerful tool to perform motion correction for two-photon Ca2+ imaging data, which may facilitate online imaging experiments in the future.

Compartmentalized dendritic plasticity during associative learning.

Experience-dependent changes in behavior are mediated by long-term functional modifications in brain circuits. Activity-dependent plasticity of synaptic input is a major underlying cellular process. Although we have a detailed understanding of synaptic and dendritic plasticity in vitro, little is known about the functional and plastic properties of active dendrites in behaving animals. Using deep brain two-photon Ca2+ imaging, we investigated how sensory responses in amygdala principal neurons develop upon classical fear conditioning, a form of associative learning. Fear conditioning induced differential plasticity in dendrites and somas regulated by compartment-specific inhibition. Our results indicate that learning-induced plasticity can be uncoupled between soma and dendrites, reflecting distinct synaptic and microcircuit-level mechanisms that increase the computational capacity of amygdala circuits.

Brain-wide projection reconstruction of single functionally defined neurons

Reconstructing axonal projections of single neurons at the whole-brain level is currently a converging goal of the neuroscience community that is fundamental for understanding the logic of information flow in the brain. Thousands of single neurons from different brain regions have recently been morphologically reconstructed, but the corresponding physiological functional features of these reconstructed neurons are unclear. By combining two-photon Ca2+ imaging with targeted single-cell plasmid electroporation, we reconstruct the brain-wide morphologies of single neurons that are defined by a sound-evoked response map in the auditory cortices (AUDs) of awake mice. Long-range interhemispheric projections can be reliably labelled via co-injection with an adeno-associated virus, which enables enhanced expression of indicator protein in the targeted neurons. Here we show that this method avoids the randomness and ambiguity of conventional methods of neuronal morphological reconstruction, offering an avenue for developing a precise one-to-one map of neuronal projection patterns and physiological functional features.