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

Abstract

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 ...

In vivo imaging of neural activity

“…Finally, other deep-brain imaging strategies involve the physical insertion into the brain of a gradient-index (GRIN) lens to directly access a deep layer 84 , or of a microprism, which provides views of a vertical cross section of the brain 85,86 (Fig. 5c).…”

Distinct speed dependence of entorhinal island and ocean cells, including respective grid cells

“…Awake in vivo calcium imaging has been implemented previously in EC (16,17), but thus far, only in head-fixed virtual linear track systems, without identification of neural type. We overcame the technical difficulty of directly and separately monitoring the activity of island and ocean cells from freely behaving mice in a 2D open space by combining a cell type-specific GCaMP-labeling method with the use of a miniaturized endoscope.…”

Cellular Level Brain Imaging in Behaving Mammals: An Engineering Approach

“…It is also relatively straightforward to perform two-color fluorescence imaging, such as for distinguishing within a broad population of indicator-labeled cells the activity of a dual-labeled subset, defined genetically or by its connectivity (Chen et al, 2013b; Inoue et al, 2015). With the help of microendoscopes or microprisms that can be inserted into the live brain, a variety of viewing angles and deep brain areas can be optically accessed that would be otherwise prohibitive (Andermann et al, 2013; Barretto et al, 2009, 2011; Chia and Levene, 2009; Heys et al, 2014; Jung et al, 2004; Jung and Schnitzer, 2003; Levene et al, 2004; Low et al, 2014). Head fixation also eases certain auxiliary manipulations, such as a mid-session intracranial drug delivery or visually guided electrical recording (Lovett-Barron et al, 2014; Nimmerjahn et al, 2009).…”

“…However, the combination of the integrated microscope and various micro-endoscopes (typically 1,000–500 μm in diameter; up to ~8 mm long) has proven surprisingly versatile in examining brain areas never before imaged optically in live mammals (Jennings et al, 2015). By adding a microprism to the tip of the microendoscope to form a periscope probe (Murayama and Larkum, 2009; Murayama et al, 2007, 2009), or using a microprism in direct combination with the microscope (Andermann et al, 2013; Chia and Levene, 2009; Heys et al, 2014; Low et al, 2014), still more brain areas and anatomical views will be accessible in freely behaving mice.…”