Olga Garaschuk

In vivo two-photon calcium imaging of neuronal networks

Two-photon calcium imaging is a powerful means for monitoring the activity of distinct neurons in brain tissue in vivo. In the mammalian brain, such imaging studies have been restricted largely to calcium recordings from neurons that were individually dye-loaded through microelectrodes. Previous attempts to use membrane-permeant forms of fluorometric calcium indicators to load populations of neurons have yielded satisfactory results only in cell cultures or in slices of immature brain tissue. Here we introduce a versatile approach for loading membrane-permeant fluorescent indicator dyes in large populations of cells. We established a pressure ejection-based local dye delivery protocol that can be used for a large spectrum of membrane-permeant indicator dyes, including calcium green-1 acetoxymethyl (AM) ester, Fura-2 AM, Fluo-4 AM, and Indo-1 AM. We applied this dye-loading protocol successfully in mouse brain tissue at any developmental stage from newborn to adult in vivo and in vitro. In vivo two-photon Ca2+ recordings, obtained by imaging through the intact skull, indicated that whisker deflection-evoked Ca2+ transients occur in a subset of layer 2/3 neurons of the barrel cortex. Thus, our results demonstrate the suitability of this technique for real-time analyses of intact neuronal circuits with the resolution of individual cells.

Clusters of Hyperactive Neurons Near Amyloid Plaques in a Mouse Model of Alzheimer's Disease

The neurodegeneration observed in Alzheimers disease has been associated with synaptic dismantling and progressive decrease in neuronal activity. We tested this hypothesis in vivo by using two-photon Ca2+ imaging in a mouse model of Alzheimers disease. Although a decrease in neuronal activity was seen in 29% of layer 2/3 cortical neurons, 21% of neurons displayed an unexpected increase in the frequency of spontaneous Ca2+ transients. These “hyperactive” neurons were found exclusively near the plaques of amyloid β–depositing mice. The hyperactivity appeared to be due to a relative decrease in synaptic inhibition. Thus, we suggest that a redistribution of synaptic drive between silent and hyperactive neurons, rather than an overall decrease in synaptic activity, provides a mechanism for the disturbed cortical function in Alzheimers disease.

Large-scale oscillatory calcium waves in the immature cortex

Two-photon imaging of large neuronal networks in cortical slices of newborn rats revealed synchronized oscillations in intracellular Ca2+ concentration. These spontaneous Ca2+ waves usually started in the posterior cortex and propagated slowly (2.1 mm per second) toward its anterior end. Ca2+ waves were associated with field-potential changes and required activation of AMPA and NMDA receptors. Although GABAA receptors were not involved in wave initiation, the developmental transition of GABAergic transmission from depolarizing to hyperpolarizing (around postnatal day 7) stopped the oscillatory activity. Thus we identified a type of large-scale Ca2+ wave that may regulate long-distance wiring in the immature cortex.

Targeted bulk-loading of fluorescent indicators for two-photon brain imaging in vivo

One of the challenges for modern neuroscience is to understand the rules of concerted neuronal function in vivo. This question can be addressed using noninvasive high-resolution imaging techniques like two-photon microscopy. This protocol describes a versatile approach for in vivo two-photon calcium imaging of neural networks, stained with membrane-permeant fluorescent-indicator dyes. It is based on a targeted pressure ejection of the dye into the tissue of interest and can be used for a large spectrum of indicator dyes, including Oregon Green 488 BAPTA-1 acetoxymethyl ester and Fura-2 acetoxymethyl ester. Through the use of dye mixtures and multicolor imaging, this technique allows the visualization of distinct neurons and glial cells up to 500 μm below the brain surface. It is suitable for staining the brain tissue of various different species (e.g., mouse, rat, cat and zebrafish) at all developmental stages. When combined with brain microendoscopy, it allows the monitoring of intracellular calcium signals in awake, behaving animals. The total time required to carry out the protocol, including dissection and cell staining, is ∼2 h. Thereafter, imaging experiments might be performed for at least 6 h.

Cortical calcium waves in resting newborn mice

Using a new optical fiber–based approach, we demonstrate the presence of recurrent Ca2+ transients in cortical neurons of non-anesthetized newborn mice in vivo. These Ca2+ waves reflect the correlated activity of thousands of cells and were detected only in resting, but not in moving pups. Our results suggest that Ca2+-dependent cortical maturation occurs predominantly during the intermittent sleep-like resting periods that are characteristic for the first days of postnatal life.

Improved calcium imaging in transgenic mice expressing a troponin C-based biosensor.

Fluorescent Ca2+ indicator proteins (FCIPs) are attractive tools for studying Ca2+ dynamics in live cells. Here we describe transgenic mouse lines expressing a troponin C (TnC)-based biosensor. The biosensor is widely expressed in neurons and has improved Ca2+ sensitivity both in vitro and in vivo. This allows FCIP-based two-photon Ca2+ imaging of distinct neurons and their dendrites in vivo, and opens a new avenue for structure-function analysis of intact neuronal circuits.

Brain tumour cells interconnect to a functional and resistant network

Astrocytic brain tumours, including glioblastomas, are incurable neoplasms characterized by diffusely infiltrative growth. Here we show that many tumour cells in astrocytomas extend ultra-long membrane protrusions, and use these distinct tumour microtubes as routes for brain invasion, proliferation, and to interconnect over long distances. The resulting network allows multicellular communication through microtube-associated gap junctions. When damage to the network occurred, tumour microtubes were used for repair. Moreover, the microtube-connected astrocytoma cells, but not those remaining unconnected throughout tumour progression, were protected from cell death inflicted by radiotherapy. The neuronal growth-associated protein 43 was important for microtube formation and function, and drove microtube-dependent tumour cell invasion, proliferation, interconnection, and radioresistance. Oligodendroglial brain tumours were deficient in this mechanism. In summary, astrocytomas can develop functional multicellular network structures. Disconnection of astrocytoma cells by targeting their tumour microtubes emerges as a new principle to reduce the treatment resistance of this disease.

Sparsification of neuronal activity in the visual cortex at eye-opening

Eye-opening represents a turning point in the function of the visual cortex. Before eye-opening, the visual cortex is largely devoid of sensory inputs and neuronal activities are generated intrinsically. After eye-opening, the cortex starts to integrate visual information. Here we used in vivo two-photon calcium imaging to explore the developmental changes of the mouse visual cortex by analyzing the ongoing spontaneous activity. We found that before eye-opening, the activity of layer 2/3 neurons consists predominantly of slow wave oscillations. These waves were first detected at postnatal day 8 (P8). Their initial very low frequency (0.01 Hz) gradually increased during development to ≈0.5 Hz in adults. Before eye-opening, a large fraction of neurons (>75%) was active during each wave. One day after eye-opening, this dense mode of recruitment changed to a sparse mode with only 36% of active neurons per wave. This was followed by a progressive decrease during the following weeks, reaching 12% of active neurons per wave in adults. The possible role of visual experience for this process of sparsification was investigated by analyzing dark-reared mice. We found that sparsification also occurred in these mice, but that the switch from a dense to a sparse activity pattern was delayed by 3–4 days as compared with normally-reared mice. These results reveal a modulatory contribution of visual experience during the first days after eye-opening, but an overall dominating role of intrinsic factors. We propose that the transformation in network activity from dense to sparse is a prerequisite for the changed cortical function at eye-opening.

Microglial repopulation model reveals a robust homeostatic process for replacing CNS myeloid cells

Under most physiological circumstances, monocytes are excluded from parenchymal CNS tissues. When widespread monocyte entry occurs, their numbers decrease shortly after engraftment in the presence of microglia. However, some disease processes lead to focal and selective loss, or dysfunction, of microglia, and microglial senescence typifies the aged brain. In this regard, the long-term engraftment of monocytes in the microglia-depleted brain remains unknown. Here, we report a model in which a niche for myeloid cells was created through microglia depletion. We show that microglia-depleted brain regions of CD11b-HSVTK transgenic mice are repopulated with new Iba-1-positive cells within 2 wk. The engrafted cells expressed high levels of CD45 and CCR2 and appeared in a wave-like pattern frequently associated with blood vessels, suggesting the engrafted cells were peripheral monocytes. Although two times more numerous and morphologically distinct from resident microglia up to 27 wk after initial engraftment, the overall distribution of the engrafted cells was remarkably similar to that of microglia. Two-photon in vivo imaging revealed that the engrafted myeloid cells extended their processes toward an ATP source and displayed intracellular calcium transients. Moreover, the engrafted cells migrated toward areas of kainic acid-induced neuronal death. These data provide evidence that circulating monocytes have the potential to occupy the adult CNS myeloid niche normally inhabited by microglia and identify a strong homeostatic drive to maintain the myeloid component in the mature brain.

Optical monitoring of brain function in vivo: from neurons to networks

The precise understanding of the cellular and molecular basis of brain function requires the direct assessment of the activity of defined cells in vivo. A promising approach for such analyses is two-photon microscopy in combination with appropriate cell labeling techniques. Here, we review the multi-cell bolus loading (MCBL) method that involves the use of membrane-permeant fluorescent indicator dyes. We show that this approach is useful for the functional analysis of clusters of neurons and glial cells in vivo. Work from our and other laboratories shows that the techniques that were previously feasible only in brain slices, like targeted patch clamp recordings from identified cells or pharmacological manipulations in confined brain regions, can now be used also in vivo. We also show that MCBL and two-photon imaging can be easily combined with other labeling techniques, particularly with those involving the use of genetically encoded, green-fluorescent-protein-based indicators. Finally, we examine recent applications of MCBL/two-photon imaging for the analysis of various brain regions, including the somatosensory and the visual cortex.