Imaging In Awake Mice Research Articles

Binocular integration of retinal motion information underlies optic flow processing by the cortex

SummaryLocomotion creates various patterns of optic flow on the retina, which provide the observer with information about their movement relative to the environment. However, it is unclear how these optic flow patterns are encoded by the cortex. Here, we use two-photon calcium imaging in awake mice to systematically map monocular and binocular responses to horizontal motion in four areas of the visual cortex. We find that neurons selective to translational or rotational optic flow are abundant in higher visual areas, whereas neurons suppressed by binocular motion are more common in the primary visual cortex. Disruption of retinal direction selectivity in Frmd7 mutant mice reduces the number of translation-selective neurons in the primary visual cortex and translation- and rotation-selective neurons as well as binocular direction-selective neurons in the rostrolateral and anterior visual cortex, blurring the functional distinction between primary and higher visual areas. Thus, optic flow representations in specific areas of the visual cortex rely on binocular integration of motion information from the retina.

CSIG-18. CALCIUM COMMUNICATION IN GLIOMA: CRUCIAL PACEMAKER CELLS GOVERN TUMOR PROGRESSION

Abstract BACKGROUND Gliomas form therapy-resistant multicellular networks, using neurite-like protrusions (called Tumor Microtubes [TMs]), and display intercellular calcium transients, that drive brain tumor malignancy. METHODS A refined in vitro model was established to study calcium communication and its molecular toolkit in patient-derived glioma cells. In vitro results were then validated with longitudinal in vivo two-photon calcium imaging in awake mice. RESULTS Here we first describe the discovery of a small subpopulation of glioma cells that display intrinsically active calcium oscillations and behave like pacemakers. Accordingly, these pacemaker-like glioma cells trigger synchronized calcium activity in tumor cells that are connected with them via TMs. The application of network theory shows that glioma cell networks follow a scale-free and small-world topology, governed by a subpopulation of highly connected hub cells. Interestingly, the pacemaker cells most often act as these hubs, hence communicating with many other network members. Graph theory predicts that such network design displays a high degree of robustness, making it resistant to random damage as seen after radio- and chemotherapy – but extremely vulnerable to selective damage to key hubs. In line with this theory, laser ablation of the pacemaker-like hub cells splintered the malignant tumor network into small isolated cell clusters and led to increased cell death, whereas laser ablation of random tumor cells showed no effect. Among others, KCNN4 channels, which are upregulated in gliomas, were identified as drivers of the pacemaker-like hub cells. Suppressing their pacemaking-abilities by KCNN4 inhibition strongly reduced calcium communication and tumor growth. CONCLUSION In summary, coordinated calcium communication in glioma drives brain tumor malignancy. A specific tumor cell subpopulation was identified that acts as hubs of the multicellular network and initiates global calcium activity. The network’s vulnerability to loosing these pacemaker-like hub cells suggests a novel targeted approach against the resistant networks of gliomas.

Seizures elevate gliovascular unit Ca2+ and cause sustained vasoconstriction.

Seizures can result in a severe hypoperfusion/hypoxic attack that causes postictal memory and behavioral impairments. However, neither postictal changes to microvasculature nor Ca2+ changes in key cell types controlling blood perfusion have been visualized in vivo, leaving essential components of the underlying cellular mechanisms unclear. Here, we use 2-photon microvascular and Ca2+ imaging in awake mice to show that seizures result in a robust vasoconstriction of cortical penetrating arterioles, which temporally mirrors the prolonged postictal hypoxia. The vascular effect was dependent on cyclooxygenase 2, as pretreatment with ibuprofen prevented postictal vasoconstriction. Moreover, seizures caused a rapid elevation in astrocyte endfoot Ca2+ that was confined to the seizure period, and vascular smooth muscle cells displayed a significant increase in Ca2+ both during and following seizures, lasting up to 75 minutes. Our data show enduring postictal vasoconstriction and temporal activities of 2 cell types within the neurovascular unit that are associated with seizure-induced hypoperfusion/hypoxia. These findings support prevention of this event may be a novel and tractable treatment strategy in patients with epilepsy who experience extended postseizure impairments.

An ultrasensitive biosensor for high-resolution kinase activity imaging in awake mice.

Protein kinases control nearly every facet of cellular function. These key signaling nodes integrate diverse pathway inputs to regulate complex physiological processes, and aberrant kinase signaling is linked to numerous pathologies. While fluorescent protein-based biosensors have revolutionized the study of kinase signaling by allowing direct, spatiotemporally precise kinase activity measurements in living cells, powerful new molecular tools capable of robustly tracking kinase activity dynamics across diverse experimental contexts are needed to fully dissect the role of kinase signaling in physiology and disease. Here, we report the development of an ultrasensitive, second-generation excitation-ratiometric protein kinase A (PKA) activity reporter (ExRai-AKAR2), obtained via high-throughput linker library screening, that enables sensitive and rapid monitoring of live-cell PKA activity across multiple fluorescence detection modalities, including plate reading, cell sorting and one- or two-photon imaging. Notably, in vivo visual cortex imaging in awake mice reveals highly dynamic neuronal PKA activity rapidly recruited by forced locomotion.

Tracking Calcium Dynamics and Immune Surveillance at the Choroid Plexus Blood-Cerebrospinal Fluid Interface

The choroid plexus (ChP) epithelium is a source of secreted signaling factors in cerebrospinal fluid (CSF) and a key barrier between blood and brain. Here, we develop imaging tools to interrogate these functions in adult lateral ventricle ChP in whole-mount explants and in awake mice. By imaging epithelial cells in intact ChP explants, we observed calcium activity and secretory events that increased in frequency following delivery of serotonergic agonists. Using chronic two-photon imaging in awake mice, we observed spontaneous subcellular calcium events as well as strong agonist-evoked calcium activation and cytoplasmic secretion into CSF. Three-dimensional imaging of motility and mobility of multiple types of ChP immune cells at baseline and following immune challenge or focal injury revealed a range of surveillance and defensive behaviors. Together, these tools should help illuminate the diverse functions of this understudied body-brain interface.

Brain-wide structural and functional disruption in mice with oligodendrocyte-specific Nf1 deletion is rescued by inhibition of nitric oxide synthase

Neurofibromin gene (NF1) mutation causes neurofibromatosis type 1 (NF1), a disorder in which brain white matter deficits identified by neuroimaging are common, yet of unknown cellular etiology. In mice, Nf1 loss in adult oligodendrocytes causes myelin decompaction and increases oligodendrocyte nitric oxide (NO) levels. Nitric oxide synthase (NOS) inhibitors rescue this pathology. Whether oligodendrocyte pathology is sufficient to affect brain-wide structure and account for NF1 imaging findings is unknown. Here we show that Nf1 gene inactivation in adult oligodendrocytes (Plp-Nf1fl/+ mice) results in a motor coordination deficit. Magnetic resonance imaging in awake mice showed that fractional anisotropy is reduced in Plp-Nf1fl/+ corpus callosum and that interhemispheric functional connectivity in the motor cortex is also reduced, consistent with disrupted myelin integrity. Furthermore, NOS-specific inhibition rescued both measures. These results suggest that oligodendrocyte defects account for aspects of brain dysfunction in NF1 that can be identified by neuroimaging and ameliorated by NOS inhibition.

Pharmaco-fUS: Quantification of pharmacologically-induced dynamic changes in brain perfusion and connectivity by functional ultrasound imaging in awake mice

There is a critical need for reliable quantitative biomarkers to assess functional brain alterations in mouse models of neuropsychiatric diseases, but current imaging methods measuring drug effects through the neurovascular coupling, face issues including poor sensitivity, drug-induced changes in global brain perfusion and the effects of anesthesia.Here we demonstrate the proof-of-concept of a minimally-invasive fUS imaging approach to detect the acute cholinergic modulatory effects of Scopolamine (ScoP) on functional brain connectivity in awake and behaving mice, through the intact skull. A machine-learning algorithm constructed an ad-hoc pharmacological score from the ScoP-induced changes in connectivity patterns of five mice. The discrimination model shows important ScoP-induced increase of the hippocampo-cortical connectivity. The pharmacological score led to robust discrimination of ScoP treatment from baseline in an independent dataset and showed, in another independent group, dose-dependent specific effects of central cholinergic modulation of functional connectivity, independent from global brain perfusion changes. In conclusion, we introduce pharmaco-fUS as a simple, robust, specific and sensitive modality to monitor drug effects on perfusion and functional connectivity in the awake mouse brain.

An Ultrasensitive Fluorescent Biosensor for Multi‐modal Kinase Activity Detection and High‐resolution Imaging in Awake Mice

Fluorescent protein (FP)‐based kinase activity biosensors are powerful tools for probing the spatiotemporal dynamics of signaling pathways in living cells. Kinase activity imaging is frequently used to unravel the molecular regulation of Protein Kinase A (PKA), a prototypical kinase that integrates multiple pathways to orchestrate diverse physiological processes. Yet the limited sensitivity of most kinase biosensors restricts their reliable application in physiologically relevant in vivo systems or clinically useful high‐content detection modalities. We previously developed an excitation‐ratiometric PKA activity reporter (ExRai‐AKAR) which utilizes a PKA substrate peptide and phosphoamino acid‐binding forkhead‐associated 1 (FHA1) domain to modulate the peak excitation wavelength of cpEGFP in response to phosphorylation by PKA. Here, we report the further development of a new, ultrasensitive excitation‐ratiometric PKA activity reporter (ExRai‐AKAR2) obtained via high‐throughput linker library screening. This powerful and versatile molecular tool enables sensitive and rapid monitoring of live‐cell PKA activity across multiple fluorescence detection modalities, including plate reading, cell sorting, and one‐ or two‐photon imaging. In vivo visual cortex imaging in awake mice reveals highly dynamic neuronal PKA activity rapidly recruited by forced locomotion. The performance of ExRai‐AKAR2 promises to successfully expand kinase activity imaging beyond the petri dish.Support or Funding InformationThis work was supported by Brain Initiative Grant R01 MH111516 and by R35 CA197622, R01 DK073368, and R01 GM111665 (to J.Z.).

Synaptic Zinc Enhances Inhibition Mediated by Somatostatin, but not Parvalbumin, Cells in Mouse Auditory Cortex

Cortical inhibition is essential for brain activity and behavior. Yet, the mechanisms that modulate cortical inhibition and their impact on sensory processing remain less understood. Synaptically released zinc, a neuromodulator released by cortical glutamatergic synaptic vesicles, has emerged as a powerful modulator of sensory processing and behavior. Despite the puzzling finding that the vesicular zinc transporter (ZnT3) mRNA is expressed in cortical inhibitory interneurons, the actions of synaptic zinc in cortical inhibitory neurotransmission remain unknown. Using in vitro electrophysiology and optogenetics in mouse brain slices containing the layer 2/3 (L2/3) of auditory cortex, we discovered that synaptic zinc increases the quantal size of inhibitory GABAergic neurotransmission mediated by somatostatin (SOM)- but not parvalbumin (PV)-expressing neurons. Using two-photon imaging in awake mice, we showed that synaptic zinc is required for the effects of SOM- but not PV-mediated inhibition on frequency tuning of principal neurons. Thus, cell-specific zinc modulation of cortical inhibition regulates frequency tuning.

Synaptic Mechanisms Underlying the Network State-Dependent Recruitment of VIP-Expressing Interneurons in the CA1 Hippocampus.

Disinhibition is a widespread circuit mechanism for information selection and transfer. In the hippocampus, disinhibition of principal cells is provided by the interneuron-specific interneurons that express the vasoactive intestinal polypeptide (VIP-IS) and innervate selectively inhibitory interneurons. By combining optophysiological experiments with computational models, we determined the impact of synaptic inputs onto the network state-dependent recruitment of VIP-IS cells. We found that VIP-IS cells fire spikes in response to both the Schaffer collateral and the temporoammonic pathway activation. Moreover, by integrating their intrinsic and synaptic properties into computational models, we predicted recruitment of these cells between the rising phase and peak of theta oscillation and during ripples. Two-photon Ca2+-imaging in awake mice supported in part the theoretical predictions, revealing a significant speed modulation of VIP-IS cells and their preferential albeit delayed recruitment during theta-run epochs, with estimated firing at the rising phase and peak of the theta cycle. However, it also uncovered that VIP-IS cells are not activated during ripples. Thus, given the preferential theta-modulated firing of VIP-IS cells in awake hippocampus, we postulate that these cells may be important for information gating during spatial navigation and memory encoding.

Experience-Dependent Development and Maintenance of Binocular Neurons in the Mouse Visual Cortex

SUMMARYThe development of neuronal circuits requires both hard-wired gene expression and experience-dependent plasticity. Sensory processing, such as binocular vision, is especially sensitive to perturbations of experience. We investigated the experience-dependent development of the binocular visual cortex at single-cell resolution by using two-photon calcium imaging in awake mice. At eye-opening, the majority of visually responsive neurons are monocular. Binocular neurons emerge later with visual experience and acquire distinct visual response properties. Surprisingly, rather than mirroring the effects of visual deprivation, mice that lack the plasticity gene Arc show increased numbers of binocular neurons and a shift in ocular dominance during development. Strikingly, acutely removing Arc in the adult binocular visual cortex also increases the number of binocular neurons, suggesting that the maintenance of binocular circuits requires ongoing plasticity. Thus, experience-dependent plasticity is critical for the development and maintenance of circuits required to process binocular vision.

Tracking Calcium Dynamics and Immune Surveillance at the Choroid Plexus Blood-Cerebrospinal Fluid Interface

The choroid plexus (ChP) epithelium is a source of secreted signaling factors in cerebrospinal fluid (CSF) and a key barrier between blood and brain. Here, we develop imaging tools to interrogate these functions in adult lateral ventricle ChP in wholemount explants and in awake mice. By imaging epithelial cells in intact ChP explants, we observed calcium activity and secretory events that increased in frequency following delivery of serotonergic agonists. Using chronic two-photon imaging in awake mice, we observed spontaneous subcellular calcium events as well as strong agonist-evoked calcium activation and cytoplasmic secretion into CSF. Three-dimensional imaging of motility and mobility of multiple types of ChP immune cells at baseline and following immune challenge or focal injury revealed a range of surveillance and defensive behaviors. Together, these tools should help illuminate the diverse functions of this understudied body-brain interface.

Somatic and Dendritic Encoding of Spatial Variables in Retrosplenial Cortex Differs during 2D Navigation

Active amplification of organized synaptic inputs in dendrites can endow individual neurons with the ability to perform complex computations. However, whether dendrites in behaving animals perform independent local computations is not known. Such activity may be particularly important for complex behavior, where neurons integrate multiple streams of information. Head-restrained imaging has yielded important insights into cellular and circuit function, but this approach limits behavior and the underlying computations. We describe a method for full-featured 2-photon imaging in awake mice during free locomotion with volitional head rotation. We examine head direction and position encoding in simultaneously imaged apical tuft dendrites and their respective cell bodies in retrosplenial cortex, an area that encodes multi-modal navigational information. Activity in dendrites was not determined solely by somatic activity but reflected distinct navigational variables, fulfilling the requirements for dendritic computation. Our approach provides a foundation for studying sub-cellular processes during complex behaviors.

Kilohertz two-photon brain imaging in awake mice.

Two-photon microscopy is a mainstay technique for imaging in scattering media and normally provides frame-acquisition rates of ~10–30 Hz. To track high-speed phenomena, we created a two-photon microscope with 400 illumination beams that collectively sample 95,000–211,000 μm2 areas at rates up to 1 kHz. Using this microscope, we visualized microcirculatory flow, fast venous constrictions, and neuronal Ca2+ spiking with millisecond-scale timing resolution in the brains of awake mice.

Tonotopic and non-auditory organization of the mouse dorsal inferior colliculus revealed by two-photon imaging.

The dorsal (DCIC) and lateral cortices (LCIC) of the inferior colliculus are major targets of the auditory and non-auditory cortical areas, suggesting a role in complex multimodal information processing. However, relatively little is known about their functional organization. We utilized in vivo two-photon Ca2+ imaging in awake mice expressing GCaMP6s in GABAergic or non-GABAergic neurons in the IC to investigate their spatial organization. We found different classes of temporal responses, which we confirmed with simultaneous juxtacellular electrophysiology. Both GABAergic and non-GABAergic neurons showed spatial microheterogeneity in their temporal responses. In contrast, a robust, double rostromedial-caudolateral gradient of frequency tuning was conserved between the two groups, and even among the subclasses. This, together with the existence of a subset of neurons sensitive to spontaneous movements, provides functional evidence for redefining the border between DCIC and LCIC.

Disruption of NMDAR Function Prevents Normal Experience-Dependent Homeostatic Synaptic Plasticity in Mouse Primary Visual Cortex.

Homeostatic regulation of synaptic strength allows for maintenance of neural activity within a dynamic range for proper circuit function. There are largely two distinct modes of synaptic plasticity that allow for homeostatic adaptation of cortical circuits: synaptic scaling and sliding threshold (BCM theory). Previous findings suggest that the induction of synaptic scaling is not prevented by blocking NMDARs, whereas the sliding threshold model posits that the synaptic modification threshold of LTP and LTD readjusts with activity and thus the outcome of synaptic plasticity is NMDAR dependent. Although synaptic scaling and sliding threshold have been considered two distinct mechanisms, there are indications from recent studies that these two modes of homeostatic plasticity may interact or that they may operate under two distinct activity regimes. Here, we report using both sexes of mouse that acute genetic knock-out of the obligatory subunit of NMDAR or acute pharmacological block of NMDAR prevents experience-dependent homeostatic regulation of AMPAR-mediated miniature EPSCs in layer 2/3 of visual cortex. This was not due to gross changes in postsynaptic neuronal activity with inhibiting NMDAR function as determine by c-Fos expression and two-photon Ca2+ imaging in awake mice. Our results suggest that experience-dependent homeostatic regulation of intact cortical circuits is mediated by NMDAR-dependent plasticity mechanisms, which supports a sliding threshold model of homeostatic adaptation.SIGNIFICANCE STATEMENT Prolonged changes in sensory experience lead to homeostatic adaptation of excitatory synaptic strength in sensory cortices. Both sliding threshold and synaptic scaling models can account for the observed homeostatic synaptic plasticity. Here we report that visual experience-dependent homeostatic plasticity of excitatory synapses observed in superficial layers of visual cortex is dependent on NMDAR function. In particular, both strengthening of synapses induced by visual deprivation and the subsequent weakening by reinstatement of visual experience were prevented in the absence of functional NMDARs. Our results suggest that sensory experience-dependent homeostatic adaptation depends on NMDARs, which supports the sliding threshold model of plasticity and input-specific homeostatic control observed in vivo.

Pure tones modulate the representation of orientation and direction in the primary visual cortex.

Multimodal sensory integration facilitates the generation of a unified and coherent perception of the environment. It is now well established that unimodal sensory perceptions, such as vision, are improved in multisensory contexts. Whereas multimodal integration is primarily performed by dedicated multisensory brain regions such as the association cortices or the superior colliculus, recent studies have shown that multisensory interactions also occur in primary sensory cortices. In particular, sounds were shown to modulate the responses of neurons located in layers 2/3 (L2/3) of the mouse primary visual cortex (V1). Yet, the net effect of sound modulation at the V1 population level remained unclear. In the present study, we performed two-photon calcium imaging in awake mice to compare the representation of the orientation and the direction of drifting gratings by V1 L2/3 neurons in unimodal (visual only) or multimodal (audiovisual) conditions. We found that sound modulation depended on the tuning properties (orientation and direction selectivity) and response amplitudes of V1 L2/3 neurons. Sounds potentiated the responses of neurons that were highly tuned to the cue's orientation and direction but weakly active in the unimodal context, following the principle of inverse effectiveness of multimodal integration. Moreover, sound suppressed the responses of neurons untuned for the orientation and/or the direction of the visual cue. Altogether, sound modulation improved the representation of the orientation and direction of the visual stimulus in V1 L2/3. Namely, visual stimuli presented with auditory stimuli recruited a neuronal population better tuned to the visual stimulus orientation and direction than when presented alone. NEW & NOTEWORTHY The primary visual cortex (V1) receives direct inputs from the primary auditory cortex. Yet, the impact of sounds on visual processing in V1 remains controverted. We show that the modulation by pure tones of V1 visual responses depends on the orientation selectivity, direction selectivity, and response amplitudes of V1 neurons. Hence, audiovisual stimuli recruit a population of V1 neurons better tuned to the orientation and direction of the visual stimulus than unimodal visual stimuli.

In vivo imaging reveals reduced activity of neuronal circuits in a mouse tauopathy model.

Pathological alterations of tau protein play a significant role in the emergence and progression of neurodegenerative disorders. Tauopathies are characterized by detachment of the tau protein from neuronal microtubules, and its subsequent aberrant hyperphosphorylation, aggregation and cellular distribution. The exact nature of tau protein species causing neuronal malfunction and degeneration is still unknown. In the present study, we used mice transgenic for human tau with the frontotemporal dementia with parkinsonism-associated P301S mutation. These mice are prone to develop fibrillar tau inclusions, especially in the spinal cord and brainstem. At the same time, cortical neurons are not as strongly affected by fibrillar tau forms, but rather by soluble tau forms. We took advantage of the possibility to induce formation of neurofibrillary tangles in a subset of these cortical neurons by local injection of preformed synthetic tau fibrils. By using chronic in vivo two-photon calcium imaging in awake mice, we were able for the first time to follow the activity of individual tangle-bearing neurons and compare it to the activity of tangle-free neurons over the disease course. Our results revealed strong reduction of calcium transient frequency in layer 2/3 cortical neurons of P301S mice, independent of neurofibrillary tangle presence. These results clearly point to the impairing role of soluble, mutated tau protein species present in the majority of the neurons investigated in this study.

Opposing Roles of apolipoprotein E in aging and neurodegeneration.

Apolipoprotein E (APOE) effects on brain function remain controversial. Removal of APOE not only impairs cognitive functions but also reduces neuritic amyloid plaques in mouse models of Alzheimer's disease (AD). Can APOE simultaneously protect and impair neural circuits? Here, we dissociated the role of APOE in AD versus aging to determine its effects on neuronal function and synaptic integrity. Using two-photon calcium imaging in awake mice to record visually evoked responses, we found that genetic removal of APOE improved neuronal responses in adult APP/PSEN1 mice (8-10 mo). These animals also exhibited fewer neuritic plaques with less surrounding synapse loss, fewer neuritic dystrophies, and reactive glia. Surprisingly, the lack of APOE in aged mice (18-20 mo), even in the absence of amyloid, disrupted visually evoked responses. These results suggest a dissociation in APOE's role in AD versus aging: APOE may be neurotoxic during early stages of amyloid deposition, although being neuroprotective in latter stages of aging.

Superior Colliculus: A Vision for Orienting

The superior colliculus contains neurons sensitive to motion direction. New research shows that these neurons are anatomically clustered: those representing the region of visual space 'seen' by both eyes preferentially respond to nasal motion directions and others to opposite directions.