Two-photon calcium imaging of evoked activity from L5 somatosensory neurons in vivo

124

153

In vivo two-photon microscopy provides the foundation for an array of powerful techniques for optically measuring and perturbing neural circuits. However, challenging tissue properties and geometry have prevented high-resolution optical access to regions situated within deep fissures. These regions include the medial prefrontal and medial entorhinal cortex (mPFC and MEC), which are of broad scientific and clinical interest. Here, we present a method for in vivo, subcellular resolution optical access to the mPFC and MEC using microprisms inserted into the fissures. We chronically imaged the mPFC and MEC in mice running on a spherical treadmill, using two-photon laser-scanning microscopy and genetically encoded calcium indicators to measure network activity. In the MEC, we imaged grid cells, a widely studied cell type essential to memory and spatial information processing. These cells exhibited spatially modulated activity during navigation in a virtual reality environment. This method should be extendable to other brain regions situated within deep fissures, and opens up these regions for study at cellular resolution in behaving animals using a rapidly expanding palette of optical tools for perturbing and measuring network structure and function.two-photon imaging | medial prefrontal cortex | medial entorhinal cortex | grid cell O ptical tools provide powerful means to measure and perturb the structure and function of neural circuits (1). However, a key set of brain regions remains outside the reach of cellularresolution optical methods: those situated within deep fissures. In the rodent brain, these areas include the medial prefrontal and medial entorhinal cortex (mPFC and MEC), two of the most widely studied regions supporting cognitive functions. The mPFC is centrally important to planning, executive function, learning, and memory (2, 3). Understanding how the mPFC implements these functions at a mechanistic, circuit level remains a longstanding goal of the field. The entorhinal cortex forms the interface between the neocortex and hippocampus and is believed to play an essential role in episodic memory and spatial information processing. These functions are particularly associated with MEC grid cells, which fire on a hexagonal lattice as an animal moves through an environment (4). Grid cells have galvanized a large body of work investigating their computational role and the mechanisms underlying grid formation, which remain important, open questions (4, 5). Moreover, mPFC or MEC dysfunction is associated with addiction, depression, schizophrenia, Alzheimer's disease, and epilepsy.The study of the mPFC, MEC, and neighboring regions could be greatly advanced by in vivo, high-resolution optical tools. However, tissue properties and geometry present significant challenges to deploying these tools within deep fissures. For example, the depth of two-photon microscopy is limited by scattering and out-of-plane excitation (6). Access in the mammalian brain is therefore typically restricted to superficial regions (<500 ...

Section: Discussionmentioning

confidence: 99%

Cellular resolution optical access to brain regions in fissures: Imaging medial prefrontal cortex and grid cells in entorhinal cortex

Low

Tank

2014

124

153

“…The high temporal resolution may be especially useful for the analysis of highly active cells such as layer 5 excitatory pyramidal neurons (21). Finally, the range of applications of Cal-590-based Ca 2+ imaging may be enhanced when using it in combination with other deep-imaging techniques, such as regenerative amplifiers (38), adaptive optics (39), or lasers with three-photon excitation capabilities (40), extending further the potential to gain insight into neuronal network activity in regions previously inaccessible to functional two-photon imaging studies in vivo.…”

Section: Discussionmentioning

confidence: 99%

Deep two-photon brain imaging with a red-shifted fluorometric Ca ²⁺ indicator

Tischbirek

Birkner

Jia

et al. 2015

107

In vivo Ca 2+ imaging of neuronal populations in deep cortical layers has remained a major challenge, as the recording depth of two-photon microscopy is limited because of the scattering and absorption of photons in brain tissue. A possible strategy to increase the imaging depth is the use of red-shifted fluorescent dyes, as scattering of photons is reduced at long wavelengths. Here, we tested the red-shifted fluorescent Ca 2+ indicator Cal-590 for deep tissue experiments in the mouse cortex in vivo. In experiments involving bulk loading of neurons with the acetoxymethyl (AM) ester version of Cal-590, combined two-photon imaging and cell-attached recordings revealed that, despite the relatively low affinity of Cal-590 for Catransients were discernable in most neurons with a good signalto-noise ratio. Action potential-dependent Ca 2+ transients were recorded in neurons of all six layers of the cortex at depths of up to −900 μm below the pial surface. We demonstrate that Cal-590 is also suited for multicolor functional imaging experiments in combination with other Ca 2+ indicators. Ca 2+ transients in the dendrites of an individual Oregon green 1,2-bis(o-aminophenoxy)ethane-N,N,N′, N′-tetraacetic acid-1 (OGB-1)-labeled neuron and the surrounding population of Cal-590-labeled cells were recorded simultaneously on two spectrally separated detection channels. We conclude that the red-shifted Ca 2+ indicator Cal-590 is well suited for in vivo twophoton Ca 2+ imaging experiments in all layers of mouse cortex. In combination with spectrally different Ca 2+ indicators, such as OGB-1, Cal-590 can be readily used for simultaneous multicolor functional imaging experiments.calcium imaging | neuronal activity | mouse cortical circuits | multicolor functional imaging

“…In these studies, imaging was performed in barrel cortex, where L5 begins at 600 μm or more below the pial surface (14). This depth is below the reach of conventional two-photon calcium imaging, and special techniques developed to image this deep have only been used to record somatic calcium activity (19). We circumvented this issue by focusing on the whisker representation area in wM1.…”

Section: Discussionmentioning

confidence: 99%

Multibranch activity in basal and tuft dendrites during firing of layer 5 cortical neurons in vivo

Hill

Varga

Jia

et al. 2013

Layer 5 pyramidal neurons process information from multiple cortical layers to provide a major output of cortex. Because of technical limitations it has remained unclear how these cells integrate widespread synaptic inputs located in distantly separated basal and tuft dendrites. Here, we obtained in vivo twophoton calcium imaging recordings from the entire dendritic field of layer 5 motor cortex neurons. We demonstrate that during subthreshold activity, basal and tuft dendrites exhibit spatially localized, small-amplitude calcium transients reflecting afferent synaptic inputs. During action potential firing, calcium signals in basal dendrites are linearly related to spike activity, whereas calcium signals in the tuft occur unreliably. However, in both dendritic compartments, spike-associated calcium signals were uniformly distributed throughout all branches. Thus, our data support a model of widespread, multibranch integration with a direct impact by basal dendrites and only a partial contribution on output signaling by the tuft.excitatory synapses | dendritic integration | mouse motor cortex P yramidal neurons feature extensive dendritic arborizations that receive and process widespread synaptic input. Much interest has been placed on the role of individual dendritic branches in determining neuronal output (1). The single branch has been hypothesized to function as a unit of plasticity (2), protein synthesis (3), and synaptic integration (4). Moreover, single dendritic branches have been shown to generate spatially restricted regenerative events, the so-called dendritic spikes, which can process and amplify local input (5). These features raise the question of whether the input-output relation of a cortical neuron is influenced primarily by single dendrites or by multibranch activity.Layer 5 (L5) pyramidal neurons serve as the major output cell type of neocortex. Their morphology is characterized by a set of basal dendrites as well as a set of distal tuft dendrites that extend into layer 1 and are separated from the soma by a long apical trunk (6). These compartments are known to be specialized in terms of the type of synaptic input that they receive (7). Furthermore, in vitro studies of dendritic physiology have revealed several active dendritic signals in these compartments such as back-propagation of action potentials (bAPs) (8, 9), NMDA spikes resulting from clustered synaptic activation (5), and calcium spikes initiated in the apical trunk (10, 11). Despite the presence of these dendritic specializations, little is known about the importance of these features for in vivo function. Previous in vivo studies were limited by the technical challenges of recording from deep basal structures in L5 (12) or of recording multiple individual dendrites simultaneously from the same neuron (13). A complete understanding of dendritic integration in L5 pyramidal neurons requires in vivo data from both the basal and tuft compartments with single dendritic branch resolution.Here we address the question of multibranch activi...