Here, we report in vivo 3-D visualization of the layered organization of a rat olfactory bulb (OB) by a swept source optical coherence tomography (SS-OCT). The SS-OCT operates at a wavelength of 1334 nm with respective theoretical depth and lateral resolutions of 6.7 μm and 15.4 μm in air and hence it is possible to get a 3D structural map of OB in vivo at the micron level resolution with millimeter-scale imaging depth. Up until now, with methods such as MRI, confocal microscopy, OB depth structure in vivo had not been clearly visualized as these do not satisfy the criterion of simultaneously providing micron-scale spatial resolution and imaging up to a few millimeter in depth. In order to confirm the OB’s layered organization revealed by SS-OCT, we introduced the technique of electrocoagulation to make landmarks across the layered structure. To our knowledge this is such a first study that combines electrocoagulation and OCT in vivo of rat OB. Our results confirmed the layered organization of OB, and moreover the layers were clearly identified by electrocoagulation landmarks both in the OCT structural and anatomical slice images. We expect such a combined study is beneficial for both OCT and neuroscience fields.

Abstract:In rodent olfactory bulb (OB), optical intrinsic signal imaging (OISI) is commonly used to investigate functional maps to odorant stimulations. However, in such studies, the spatial resolution in depth direction (z-axis) is lost because of the integration of light from different depths. To solve this problem, we propose functional optical coherence tomography (fOCT) with periodic stimulation and continuous recording. In fOCT experiments of in vivo rat OB, propionic acid and m-cresol were used as odor stimulus presentations. Such a periodic stimulation enabled us to detect the specific odorresponses from highly scattering brain tissue. Swept source OCT operating at a wavelength of 1334 nm and a frequency of 20 kHz, was employed with theoretical depth and lateral resolutions of 6.7 μm and 15.4 μm, respectively. We succeeded in visualizing 2D cross sectional fOCT map across the neural layer structure of OCT in vivo. The detected fOCT signals corresponded to a few glomeruli of the medial and lateral parts of dorsal OB. We also obtained 3D fOCT maps, which upon integration across z-axis agreed well with OISI results. We expect such an approach to open a window for investigating and possibly addressing toward inter/intra-layer connections at high resolutions in the future. 181-192 (1997). 33. Y. Nakamichi, V. A. Kalatsky, H. Watanabe, U. M. Rajagopalan, and M. Tanifuji, "3D structure of the orientation column in cat primary cortex revealed by functional optical coherence tomography," Abstr. Soc. Neurosci. 270. 01 (2011), http://www.abstractsonline.com/Plan/ViewAbstract.aspx?sKey=d023089b-a64b-4aa9-b3cb-6129bde54e45&cKey=09b7566c-c153-4692-8113-1a04e1af0b1d&mKey=%7b8334BE29-8911-4991-8C31- 3067-3086 (2012).

Despite extensive research on primate cognitive function, understanding how anatomical connectivity at a neural circuit level relates to information transformation across different cortical areas remains primitive. New technology is needed to visualize inter-areal anatomical connectivity in living monkeys and to tie this directly to neurophysiological function. Here, we developed a novel method to investigate this structure-function relationship, by combining optical intrinsic signal imaging (OISI) with optogenetic stimulation in living monkeys (opto-OISI). The method involves expressing channelrhodophsin-2 in one area (source) followed by optical imaging of optogenetic activations in the other area (target). We successfully demonstrated the potential of the method with interhemispheric columnar projection patterns between V1/V2 border regions. Unlike the combination of optogenetics and functional magnetic resonance imaging (opto-fMRI), opto-OISI has the advantage of enabling us to detect responses of small clusters of neurons, even if the clusters are sparsely distributed. We suggest that opto-OISI can be a powerful approach to understanding cognitive function at the neural circuit level, directly linking inter-areal circuitry to fine-scale structure and function.

In studies of in vivo extracellular recording, we usually penetrate electrodes almost blindly into the neural tissue, in order to detect the neural activity from an expected target location at a certain depth. After the recording, it is necessary for us to determine the position of the electrodes precisely. Generally, to identify the position of the electrode, one method is to examine the postmortem tissue sample at micron resolution. The other method is using MRI and it does not have enough resolution to resolve the neural structures. To solve such problems, we propose swept source optical coherence tomography (SS-OCT) as a tool to visualize the cross-sectional image of the neural target structure along with the penetrating electrode. We focused on a rodent olfactory bulb (OB) as the target. We succeeded in imaging both the OB layer structure and the penetrating electrode, simultaneously. The method has the advantage of detecting the electrode shape and the position in real time, in vivo. These results indicate the possibility of using SS-OCT as a powerful tool for guiding the electrode into the target tissue precisely in real time and localizing the electrode tip during electrophysiological recordings.

Orientation tuning is a canonical neuronal response property of six-layer visual cortex that is encoded in pinwheel structures with center orientation singularities. Optical imaging of intrinsic signals enables us to map these surface two-dimensional (2D) structures, whereas lack of appropriate techniques has not allowed us to visualize depth structures of orientation coding. In the present study, we performed functional optical coherence tomography (fOCT), a technique capable of acquiring a 3D map of the intrinsic signals, to study the topology of orientation coding inside the cat visual cortex. With this technique, for the first time, we visualized columnar assemblies in orientation coding that had been predicted from electrophysiological recordings. In addition, we found that the columnar structures were largely distorted around pinwheel centers: center singularities were not rigid straight lines running perpendicularly to the cortical surface but formed twisted string-like structures inside the cortex that turned and extended horizontally through the cortex. Looping singularities were observed with their respective termini accessing the same cortical surface via clockwise and counterclockwise orientation pinwheels. These results suggest that a 3D topology of orientation coding cannot be fully anticipated from 2D surface measurements. Moreover, the findings demonstrate the utility of fOCT as an in vivo mesoscale imaging method for mapping functional response properties of cortex in the depth axis. NEW & NOTEWORTHY We used functional optical coherence tomography (fOCT) to visualize three-dimensional structure of the orientation columns with millimeter range and micrometer spatial resolution. We validated vertically elongated columnar structure in iso-orientation domains. The columnar structure was distorted around pinwheel centers. An orientation singularity formed a string with tortuous trajectories inside the cortex and connected clockwise and counterclockwise pinwheel centers in the surface orientation map. The results were confirmed by comparisons with conventional optical imaging and electrophysiological recordings.