VisualSonics White Paper:

In vivo Fiberoptic Fluorescence Microscopy in freely behaving mice

October 9, 2009 Version 1.0

Table of Contents
Introduction ......................................................................................................... 1 Experimental setup ............................................................................................... 3 Animals ............................................................................................................... 3 Materials.............................................................................................................. 3 Stereotaxic coordinates.......................................................................................... 4 Procedure ............................................................................................................ 5 Results.................................................................................................................. 6 Discussion ............................................................................................................ 8 References.......................................................................................................... 10 Supplemental Information .................................................................................. 11 Imaging wildtype mice infected with Adeno Associated Virus (AAV) ............................ 11 Appendix I: Implantation of the guide-cannula ........................................................ 14 Appendix II: Preparation of OGB1-AM .................................................................... 16

VisualSonics White Paper: In vivo Fiberoptic Fluorescence Microscopy in freely behaving mice

Introduction There are significant correlations between animal/organismic behavior and cellular processes and therefore understanding physiological and pathological brain function requires investigation at the genetic, molecular, cellular, and behavioral levels. While in vitro studies continue to provide useful information not otherwise attainable, they do not adequately reflect the complexity of the in vivo environment. Furthermore, as the tissue microenvironment plays a critical role in physiological and pathological processes alike, in situ studies which reveal neural responses in the context of intact, dynamic, and specific neural circuits are of fundamental importance (Chang JY et al 2008)). Until recently, in vivo in situ imaging of the brain has been limited to either low resolution imaging of large areas or highly invasive techniques restricted to superficial cortex. To yield powerful information of disease etiology and progression, high resolution minimally invasive imaging of deep brain in vivo and in situ is crucial. Fiberoptic fluorescence microscopy (FFM) employs optical fibers as small as 300 micrometers in diameter and offers the ability to image cellular and subcellular processes in deep brain structures including the Ventral Tegmental Area (VTA) and the substantia nigra (Sn). With FFM, structures of the deep brain can visualized for several hours making in vivo tracking of neuronal migration, cell division, promoter activity, and other relatively protracted processes amenable to study. Additionally, Davenne et al. (2005) reported that imaging throughout stereotaxic positioning of beveled microprobes into deep brain tissue showed that no cells were fractionated or distorted, as evidenced by an absence of fluorophore leakage from cells, and noted that cells appeared to slide along the bevel of the fiberoptic microprobe. Further investigation by ex vivo microscopy on brain slices following implantation of beveled microprobes revealed that tissue separation, while irremediable, was slight. Such minimally invasive access has enabled longitudinal imaging of deep brain structures over the course of several weeks (Crescent et al, unpublished results). However, studies of anesthetized models obviously lack behavioral corroboration. Furthermore, active brain states may serve to accentuate differences that only manifest partially while an animal is in the resting state (Holschneider DP and Maarek JM 2008).The flexible nature of fiberoptic microendoscopes employed for FFM and the ability to implant them into live subjects offers the ability to image freely moving animals. Simultaneous investigation of cellular and organismal behavior provides a direct and immediate means for relating causal events with consequent responses. For example, recording of neural responses to behaviorally effective deep brain stimulation (DBS) in freely moving animals provides a direct means for examining how DBS modulates the basal ganglia thalamocortical circuits and thereby improves motor function (Chang JY et al., 2008). Indeed much of our knowledge of behavioral neuroscience and our ability to relate cellular behavior to organismal behavior has been learned through electrophysiological recordings in freely moving animals. Place cells are hippocampal cells that encode spatial location. Recordings from these cells in freely moving, genetically modified mice have further advanced our understanding of how the actual cellular representation of space is influenced by genetic alterations that affect long-term potentiation (Mayford M et al. 1997). High resolution imaging offers increased confidence and deeper insight: responses of individual and small populations of neurons can be acquired, coupling morphology and function, and events such as motility and division.

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However, there have remained significant limitations to widespread implementation of FFM for study of deep brain in freely moving animals including size, functionality, image stability, and access to the technology. The size and weight needs to be small and light enough for use in mice. Recently, Flusberg et al 2008 employed the use of a 1.1g miniaturized epifluorescence microscope for imaging deep brain of freely moving mice. While this is a significant advancement over previous attempts (3.9g, Flusberg et al 2005), it remains heavy for a 25g mouse and may influence behavioral activity (particularly for studies involving ataxic mouse models). While epifluorescent microscopy enables fast imaging with large fields of view, out-of-focus light reduces image quality. With respect to image stability, sophisticated hardware, image processing software, and innovative methods for fixation of implanted fiberoptic microprobes are required to ensure that voluntary and involuntary spastic movements do not cause motion artifact in the acquired data. The Cellvizio LAB In Vivo Confocal Fluorescence Microscope overcomes these limitations and thereby provides an opportunity to longitudinally image the deep brain in situ with subcellular (3.3m) resolution, enabling unique research studies with a simultaneous correlation to behavioral performance. Importantly, Cellvizio LAB is the only commercially available solution for such sophisticated study. The Cellvizio LAB consists of a point-scanning confocal laser which improves image quality by limiting out-of-focus light while still allowing a 300m diameter field of view. Furthermore, the system is capable of 10ms frame acquisition (200 frames per second) and employs a single-pixel avalanche photodiode detector (APD) for superior temporal resolution and sensitivity, respectively. The recent development of the CerboFlex Probe and NeuroPak Deep Brain Imaging System provide a lightweight solution for chronic imaging of in situ deep brain in freely moving mice. The CerboFlex is a fiberoptic microprobe comprised of (tens of) thousands of individual step-index fiber optics encased within a single bundle 300um in diameter. Nonordered arrangement of these fibers eliminates crosstalk between adjacent fibers and maintains high contrast and image quality. The NeuroPak System employs surgical implantation of a <290mg stabilization plate which allows for a sturdy, mechanical, nonpermanent connection of the CerboFlex. Because the CerboFlex itself is not permanently implanted, it can be removed, cleaned, and recalibrated prior to every imaging session, ensuring reliable results and quantification in longitudinal studies of the same experimental animal. This note explains how researchers from the Institut Pasteur (Paris, France) used the Cellvizio LAB to image neurons in the Hippocampus (see supplemental data), Sn, and VTA in freely behaving mice.

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Experimental setup Animals Transgenic mice (Thy1-CerTN-L15; Heim et al., 2007) expressing GFP were used for imaging the substantia nigra while Th-gfp mice (stably transfected with a vector engineered to express GFP under the rat tyrosine hydroxylase promoter; Sawamoto et al., 2001) were employed for imaging the VTA. Animals were anaesthetized by intra-peritoneal injection of Ketamine/Xylazine (0.1/0.01 mg per gram of body weight). Alternatively, animals can be anesthetized using inhaled isofluorane (3% in Oxygen) which ensures continuous immobilization and allows for faster recovery. Materials NeuroPak Deep Brain Imaging System

Figure 1. The NeuroPak Deep Brain Imaging System includes the CerboFlex Probe, 6 implants and required screws, a guide holder for use with a stereotaxic device and complete procedural information for performing the implant surgery. Shown on the right is the implant itself, with the guide post and cannula. The implant weighs less than 300mg

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CerboFlex

Figure 2. The CerboFlex Probe mounted on the implant. The flexible fiber probe is shown protruding from the cannula. The depth of penetration may be accurately controlled during insertion using a stereotaxic device. The fiber is 300 microns in diameter, with a beveled tip for minimally invasive access. Cellvizio LAB imaging system (488nm excitation; VisualSonics, Toronto, Canada) QuantiKit 488 Calibration kit (VisualSonics, Toronto, Canada) Stereotaxic equipment (World Precision Instruments, Florida) ImageCell software

Stereotaxic coordinates The guide-cannula is stereotaxically inserted in the mouse brain above the targeted brain area and the CerboFlex then lowered to the anatomical target according to bregma coordinates (Paxinos and Franklin, The Mouse brain in stereotaxic coordinates; Academic Press). Coordinates used are : AP= -3,3 mm, L= 1,3 mm, Z=-3,4 mm (Substantia nigra, reticulata) and AP = -3,4 mm, L = 0,5 mm and Z =- 4,5 mm (VTA).

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Procedure The experiment required two distinct phases surgical implantation (1) and imaging (2). 1) At least one week prior the first imaging session the mouse underwent stereotaxic surgery to implant a guide-cannula into the skull above the targeted structures (See appendix II for supplemental information). 2) For imaging, mice were anesthetized and the CerboFlex imaging microprobe was stereotaxically guided to the target structures according to Z coordinates until fluorescent neurons are identified *. The CerboFlex was then mechanically secured to the guide-cannula using a screw to ensure stability throughout the duration of the imaging experiments. Animals were allowed to recover from anesthesia and images acquired for various periods of time thereafter while freely behaving in an open field cage. At the end of individual imaging experiments, animals were re-anesthetized to carefully remove the CerboFlex from the guide-cannula under stereotaxic guidance. *note that calibration steps were made according to Cellvizio LAB guidelines immediately prior to this step.

Figure 3 : Thy1-CerTN-L15 mouse with a guide-cannula implanted above the Sn.

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Results The CerboFlex microprobe was introduced into an anesthetized Th-GFP mouse through the surgically implanted guide-cannula and into the VTA under stereotaxic guidance according to Z coordinates until fluorescent neurons were identified (Figure 4a, t=0). As the animal recovered from anesthetic, spastic movements did not alter the position of the CerboFlex relative to the VTA as evidenced by the retention of the original imaging field of view acquired (Figure 4b, t=20min). Fluorescent dopaminergic neurons within the VTA were intermittently imaged for longer than one hour with no change in the field of view (Figure 4). Notably, background autofluorescence levels and depreciation of image quality due to photobleaching did not appreciably affect detection and visualization of fluorescent neurons in the field of view (Figure 4 c and d, t=55min and t=70min respectively).

Figure 4 : Individual frames of a Neuron within the VTA extracted from different sequences of images acquired at several time points. The red arrows point to a brightly fluorescent neuron in the VTA of a Th-GFP mouse. Notice that the field of view remains unchanged throughout the experiment.

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Similar results have been obtained for neurons of the Substantia nigra in Thy1-CerTN-L15 mice over a much longer period of time. In order to reduce photobleaching and maintain sensitivity, intermittent image acquisition with 100% laser intensity was limited to 10 second sequences repeated several times. As shown in Figure 5, a brightly fluorescent neuron was observed for more than 3 hours without image distortion and with minimal depreciation of image quality.

Figure 5: Images of fluorescent neurons within the Substantia nigra of a Thy1-CerTN-L15 mouse. The Substantia nigra was identified according to stereotaxic coordinates and observable fluorescence. The field of view was stable for more than 3 hours, sufficient for simultaneous study of cellular and organismal behavior.

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While these studies were carried out in transgenic animals, similar results have been obtained in the dorsal part of the mouse hippocampus (figure 9) in normal mice after injection of a modified Adeno Associated Virus (AAV) vector that induces cytoplasmic expression of GFP (See supplemental information). Discussion Owing to the confocal approach, nonordered fiberoptic bundle, and advanced algorithms of the Cellvizio LAB and CerboFlex along with the lightweight, stable and semi-permanent design of the NeuroPak, the images of fluorescent neurons in the VTA and Sn of freely moving mice was devoid of motion artifact. Image stability is crucial when one considers the cellular study of epileptic events in animals during seizure where convulsive behavior is expected. It is also important when quantifying cellular events that occur over a longer timescale such as neuronal migration. Davenne et al.(2005) elegantly quantified the velocity of neuroblasts migrating in vivo from the subventricular zone along the rostral migratory steam to the olfactory bulb in anesthetized adult mice. Changes in the number, direction, and velocity of migrating cells may be activity-dependent in response to olfactory stimuli; indeed a clear relationship has been shown between olfactory performance and the quantity of newborn neurons in the olfactory bulb (Lledo and Saghatelyan (2005); Rochefort, C. et al. (2002); Gheusi, G. et al. (2000); Enwere, E. et al. (2004). Monitoring of migrating neurons in freely moving animals and noninvasive observation of the bulbular neuronal network through the nasal cavity may serve to address the adaptive response required for fine adjustment of olfactory ability (Vincent et al. 2006). The concept of restructuring in the adult brain is, of course, not limited to the olfactory system; importantly, evidence suggests that neurogenesis occurs in the adult mammalian Sn (Zhao, M. et al. 2003). Monitoring of cellular responses in the Sn of unrestrained, awake animals is imperative for a comprehensive understanding of etiology and progression of Schizophrenia and Parkinsons disease. Equally important for this understanding is functional imaging of neuronal activity. Specific dyes (calcium or voltage sensors, such as Oregon Green Bapta-1 (OGB1)) must be injected prior the imaging session. These dyes serve as fluorescent sensors of intracellular [Ca2+], undergoing conformational changes that change the absorption/emission spectrum of the dye when in contact with Ca2+. The variation in fluorescent intensity upon Ca2+ influx, reflective of changes in neuronal activity, can be quantified using the kinetic analysis tool in the Cellvizio LAB software, ImageCell. (Note: see Appendix II for a protocol describing in vivo labeling of brain tissue with OGB1). For the experiments described in this document, animals were sacrificed by deep anesthesia to verify the precise position of the CerboFlex tip within the brain. However, due to the small diameter of the microprobe and therefore consequent restricted lesion of the brain tissue, longitudinal imaging of the same structure of the same animal can be acquired. Since these initial experiments, researchers at Institut Pasteur have successfully acquired images of the same freely moving animal over several weeks (unpublished data). While image stability throughout an individual imaging session is important, so is exact repositioning the CerboFlex for longitudinal studies. Because the position of the guide cannula is fixed relative to the anatomical structure of interest, precision when reintroducing the CerboFlex is ensured. Furthermore, in addition to the stereotaxic Z coordinates, images are acquired during the positioning of the CerboFlex which thereby provides another point of

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reference when fluorescence is restricted to specific brain areas. The Cellvizio LAB is a unique, commercially available imaging system that affords scientists the opportunity to image neurons in vivo and in situ of freely moving animals, even in the deepest parts of the brain. Due to the small diameter of the CerboFlex tip and to the flexibility of the probe, small animals can move freely in their environment or various behavioral mazes while images of the neuronal network are acquired over long periods of time in longitudinal studies of the same animal. The Cellvizio LAB In Vivo Confocal Fluorescence Microscope the CerboFlex Deep Brain Imaging Probe, and the NeuroPak Deep Brain Imaging System are distributed globally by VisualSonics and its distribution partners. For more information, please visit www.visualsonics.com

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References 1. 2. 3. Michael Eisenstein (2009) Getting inside their minds Nature Methods; 6, 773-781 Chang JY et al 2008 Enwere, E. et al. (2004) Aging results in reduced epidermal growth factor receptor signaling, diminished olfactory neurogenesis, and deficits in fine olfactory discrimination. J. Neurosci. 24, 83548365 Flusberg BA, Nimmerjahn A, Cocker ED, Mukamel EA, Barretto RP, Ko TH, Burns LD, Jung JC, Schnitzer MJ. (2008) High-speed, miniaturized fluorescence microscopy in freely moving mice. Nat Methods. Nov;5(11):935-8. Flusberg BA, Jung JC, Cocker ED, Anderson EP, Schnitzer MJ. In vivo brain imaging using a portable 3.9 gram two-photon fluorescence microendoscope. Opt Lett. 2005 Sep 1;30(17):2272-4. Gheusi, G. et al. (2000) Importance of newly generated neurons in the adult olfactory bulb for odor discrimination. PNAS 97, 18231828 Heim et al. (2007), Nature Methods, 4(2):127-129 Holschneider DP and Maarek JM 2008 Mayford M et al. 1997

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10. Paxinos and Franklin, The Mouse brain in stereotaxic coordinates; Academic Press 11. Rochefort, C. et al. (2002) Enriched odor exposure increases the number of newborn neurons in the adult olfactory bulb and improves odor memory. J. Neurosci. 22, 26792689 12. Sawamoto et al. (2001), PNAS, 98(11): 64236428 13. Stosiek C et al. (2003) 14. Tallini et al. (2006), PNAS, 103(12): 4753-58 15. Vincent et al. 2005 16. Zhao, M. et al. (2003)

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Supplemental Information Imaging wildtype mice infected with Adeno Associated Virus (AAV) Direct intracranial injection of a modified Adeno Associated Virus (AAV) vector (Figure S1) that induces cytoplasmic expression of GFP under the GcamP2 promoter enabled imaging of the dorsal hippocampus in a wildtype mouse. Prior the fixation of the headstage on the skull and guide-cannula insertion above the dorsal hippocampus, the vector was injected into the dorsal hippocampus (0.5 l / 5 min). A delay of 3 weeks before the imaging sessions was required to ensure adequate expression of GFP within cells.

Figure S1: The AAV vector used expressing GcamP2 and eGFP (From Tallini et al. 2006). As previously described, the CerboFlex was inserted into the brain of an anesthetized mouse and neurons were imaged in sequences of 10 sec length to reduce photobleaching. Image stability was observed for more than 4 hours as shown in figure S2.

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Figure S2: Numerous neurons of the dentate gyrus within the dorsal hippocampus after expression of GFP induced by injection of a modified Adeno Associated Virus (AAV) vector. Images were acquired at different time points.

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Appendix I: Implantation of the guide-cannula

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Appendix II: Preparation of OGB1-AM Solution preparation Use one vial of OGB 488 BAPTA-1AM (Oregon Green 488 BAPTA AM-1, MW 1258.07 g, available from Invitrogen #O-6807) 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. Add 4l of 20% pluronic acid (Invitrogen #P-3000MP) in DMSO Vortex for 3 mins After this step, the color of the solution should be slightly yellow Add 36L of Ca2+-free ACSF Add 1L of SR101 (2.5mM or 2mM mixed in ACSF) Vortex for about 3min Sonicate on ice for 5min If dye sits longer than 30min, sonicate again for 5min Pipette dye into centrifuge filter (Ultrafree MC, available from Fisher Scientific; UFC30GV25) Centrifuge for 30 sec Dilute 1: 10 in a solution containing (in mM): 150 NaCl, 2.5 KCl, 10 Hepes, pH 7.4. The final solution concentration is 1mM Fill pipette with approximately 8l

Practically add 3.97 microliters of DMSO pluronic corresponding to a 10 mM solution) in a vial of OGB1 and dilute the solution in 35.7 microliters of Hepes solution to obtain a 1mM solution Ejection Parameters Injection is done using a perfusion pump through a 36G stainless steel needle: 1microliter in 10 minutes (0.1l/min). Do not remove the needle for 10 minutes. Retraction should be incremental over a 5 minute period. Following retraction, wait one hour prior to imaging. Reference: Stosiek C et al. (2003)

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