Functional Imaging of Individual Cells in Deep Brain Regions of Freely-moving Mice

Jesus Pascual-Brazo1, Sarah Libbrecht1, Hedi Gharbi3, Chris Van Den Haute1, Zeger Debyser2, Veerle Baekeland11 Laboratory for Neurobiology and Gene Therapy. Department of Neurosciences. Faculty of Medicine, KU Leuven

2 Laboratory for Molecular Virology and Gene Therapy, Department of Pharmaceutical and Pharmacological Sciences. Faculty of Medicine. KU Leuven3 Mauna Kea Technologies. Paris (France)

INTRODUCTION

Imaging techniques, such as magnetic resonance imaging and positron emission tomography, have provided huge information about the structure and function of the brain during the last years but the low resolution and acquisition times limits the information that can be obtained with these techniques.

Recent efforts in protein engineering have given rise to sensitive genetically encoded calcium indicators (GECIs) and redox sensitive GFP, being now comparable to calcium sensitive dyes.

The optimization of viral vectors to express these reporter proteins in the brain opens the door to perform functional imaging of individual cells in the brain of freely-moving animals.

FIBERED CONFOCAL FLUORESCENCE MICROSCOPY

Cellvizio description. The system is composed of a laser scanning unit and a miniaturized microscope objective made of thousands fiber-optics. The light from the laser source (488 nm) is injected in every individual fiber and transported to the tissue. The emitted light is then returned to the detector by the same microfiber. The S300 and NeuroPak probes used for these experiments are minimally invasive (diameter 300 and 470 µm) and have a beveled tip.

Procedure. NeuroPak implant was fixed with dental cement just above of the brain region to be studied. AAV 2/5 GCaMP5/3 vectors (for calcium imaging) and Lentiviral roGFP vectors (for redox imaging) were injected through the implant channel. Two weeks later, the probe was inserted into the brain and screwed to the implant in the desired position under inhalatory anesthesia. After anesthesia recovery, the mice were allowed to freely move in the cage while recordings were performed.

Image processing. ImageCell® software was used to select regions of interest, to quantify the intensity of the fluorescent signal and to represent the data. Raw data of signal intensity was plotted for every time point.

C O N C L U S I O N S

• Optimized viral vector technology increased the signal/noise ratio of Fibered Fluorescence Microscopy images in the hippocampus of live mice.

• GCaMP5 allows to record calcium levels of several cells in freely-moving mice using this new technique.

• Mitochondrial redox state can be monitored in vivo using roGFP in awake mice with cellular resolution.

MOLECULAR VIROLOGY & GENE THERAPY

LEUVEN VIRAL VECTOR CORE - LVVC

NEUROBIOLOGY & GENE THERAPY

INCREASED SIGNAL/NOISE RATIO AFTER OPTIMIZATION

HIPPOCAMPUS HIGH TITERS HIPPOCAMPUS LOW TITERS HIPPOCAMPUS OPTIMIZED

MITOCHONDRIAL REDOX STATE IN LIVE MICE

Redox imaging. Lentiviral vector targeting redox sensitive protein (roGFP) to the mitochondria was designed and produced. Image acquisition revealed redox flashes of Individual cells in the hippocampus of live mice. ImageCell® software was used to record images at a frequency of 12 Hz, for quantification and representation of the intensity of the fluorescent signal. Raw data of signal intensity was plotted for every time point.

CALCIUM IMAGING OF SEVERAL CELLS IN LIVE MICE

GCAMP5 IMAGING IN THE STRIATUM

GCAMP3 IMAGING IN THE OLFACTORY BULB

Calcium Imaging of several cells in the several brain regions of freely-moving mice. Calcium sensitive protein GCaMP5 and GCaMP3 were expressed employing AAV vectors. Signal intensity was represented after recording at frequency of 12Hz.

Conventional and optimized viral vectors were stereotactically injected to express GFP in the hippocampus. Comparison of the signal/noise ratio after conventional (high and low titers) and optimized viral vectors transduction was carried out.

ACKNOWLEDGEMENTS. A plasmid for mito-roGFP was provided by S.J. Remington (University of Oregon,USA). GCaMP5 was obtained from Addgene (L. Looger). This work has been supported by IWT-SBO/060838 Brainstim, SCIL programme financing PF/10/019 and IWT-O&O JANSSEN-DEPVEGF projects.

GCAMP5 IMAGING IN THE HIPPOCAMPUS

Cellvizio®, a Fibered Confocal Fluorescence Microscope developed by Mauna Kea Technologies, allows to obtain images with high spatial (3 µm) and temporal (5 ms) resolution in deep brain regions of freely-moving mice.