VisualSonics White Paper:In vivo Fiberoptic Fluorescence Microscopy          in freely behaving mice                      ...
Table of ContentsIntroduction ...............................................................................................
IntroductionThere are significant correlations between animal/organismic behavior and cellularprocesses and therefore unde...
However, there have remained significant limitations to widespread implementation of FFMfor study of deep brain in freely ...
Experimental setupAnimalsTransgenic mice (Thy1-CerTN-L15; Heim et al., 2007) expressing GFP were used forimaging the subst...
   CerboFlexFigure 2. The CerboFlex Probe mounted on the implant. The flexible fiber probe is shownprotruding from the ca...
ProcedureThe experiment required two distinct phases – surgical implantation (1) and imaging (2).1) At least one week prio...
ResultsThe CerboFlex microprobe was introduced into an anesthetized Th-GFP mouse through thesurgically implanted guide-can...
Similar results have been obtained for neurons of the Substantia nigra in Thy1-CerTN-L15mice over a much longer period of ...
While these studies were carried out in transgenic animals, similar results have beenobtained in the dorsal part of the mo...
reference when fluorescence is restricted to specific brain areas.The Cellvizio LAB is a unique, commercially available im...
References    1.    Michael Eisenstein (2009) Getting inside their minds          Nature Methods; 6, 773-781    2.    Chan...
Supplemental InformationImaging wildtype mice infected with Adeno Associated Virus (AAV)Direct intracranial injection of a...
VisualSonics White Paper: In vivo Fiberoptic Fluorescence Microscopy in freely behaving mice   Page 12
Figure S2: Numerous neurons of the dentate gyrus within the dorsal hippocampus afterexpression of GFP induced by injection...
Appendix I: Implantation of the guide-cannulaVisualSonics White Paper: In vivo Fiberoptic Fluorescence Microscopy in freel...
VisualSonics White Paper: In vivo Fiberoptic Fluorescence Microscopy in freely behaving mice   Page 15
Appendix II: Preparation of OGB1-AMSolution preparationUse one vial of OGB 488 BAPTA-1AM (Oregon Green 488 BAPTA AM-1, MW ...
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White Paper: In vivo Fiberoptic Fluorescence Microscopy in freely behaving mice

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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).

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Transcript of "White Paper: In vivo Fiberoptic Fluorescence Microscopy in freely behaving mice"

  1. 1. VisualSonics White Paper:In vivo Fiberoptic Fluorescence Microscopy in freely behaving mice October 9, 2009 Version 1.0
  2. 2. Table of ContentsIntroduction ......................................................................................................... 1Experimental setup ............................................................................................... 3 Animals ............................................................................................................... 3 Materials.............................................................................................................. 3 Stereotaxic coordinates.......................................................................................... 4 Procedure ............................................................................................................ 5Results.................................................................................................................. 6Discussion ............................................................................................................ 8References.......................................................................................................... 10Supplemental 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 .................................................................... 16VisualSonics White Paper: In vivo Fiberoptic Fluorescence Microscopy in freely behaving mice
  3. 3. IntroductionThere are significant correlations between animal/organismic behavior and cellularprocesses and therefore understanding physiological and pathological brain functionrequires investigation at the genetic, molecular, cellular, and behavioral levels. While invitro studies continue to provide useful information not otherwise attainable, they do notadequately reflect the complexity of the in vivo environment. Furthermore, as the tissuemicroenvironment plays a critical role in physiological and pathological processes alike, insitu studies which reveal neural responses in the context of intact, dynamic, and specificneural circuits are of fundamental importance (Chang JY et al 2008)). Until recently, in vivoin situ imaging of the brain has been limited to either low resolution imaging of large areasor highly invasive techniques restricted to superficial cortex. To yield powerful informationof disease etiology and progression, high resolution minimally invasive imaging of deepbrain in vivo and in situ is crucial.Fiberoptic fluorescence microscopy (FFM) employs optical fibers as small as 300micrometers in diameter and offers the ability to image cellular and subcellular processes indeep 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 vivotracking of neuronal migration, cell division, promoter activity, and other relativelyprotracted processes amenable to study. Additionally, Davenne et al. (2005) reported thatimaging throughout stereotaxic positioning of beveled microprobes into deep brain tissueshowed that no cells were fractionated or distorted, as evidenced by an absence offluorophore leakage from cells, and noted that cells appeared to slide along the bevel of thefiberoptic microprobe. Further investigation by ex vivo microscopy on brain slices followingimplantation of beveled microprobes revealed that tissue separation, while irremediable,was slight. Such minimally invasive access has enabled longitudinal imaging of deep brainstructures 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 manifestpartially while an animal is in the resting state (Holschneider DP and Maarek JM 2008).Theflexible nature of fiberoptic microendoscopes employed for FFM and the ability to implantthem into live subjects offers the ability to image freely moving animals.Simultaneous investigation of cellular and organismal behavior provides a direct andimmediate means for relating causal events with consequent responses. For example,recording of neural responses to behaviorally effective deep brain stimulation (DBS) infreely moving animals provides a direct means for examining how DBS modulates the basalganglia thalamocortical circuits and thereby improves motor function (Chang JY et al.,2008). Indeed much of our knowledge of behavioral neuroscience and our ability to relatecellular behavior to organismal behavior has been learned through electrophysiologicalrecordings in freely moving animals.Place cells are hippocampal cells that encode spatial location. Recordings from these cells infreely moving, genetically modified mice have further advanced our understanding of howthe actual cellular representation of space is influenced by genetic alterations that affectlong-term potentiation (Mayford M et al. 1997). High resolution imaging offers increasedconfidence and deeper insight: responses of individual and small populations of neurons canbe acquired, coupling morphology and function, and events such as motility and division.VisualSonics White Paper: In vivo Fiberoptic Fluorescence Microscopy in freely behaving mice Page 1
  4. 4. However, there have remained significant limitations to widespread implementation of FFMfor 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 foruse in mice. Recently, Flusberg et al 2008 employed the use of a 1.1g miniaturizedepifluorescence microscope for imaging deep brain of freely moving mice. While this is asignificant advancement over previous attempts (3.9g, Flusberg et al 2005), it remains‘heavy’ for a 25g mouse and may influence behavioral activity (particularly for studiesinvolving ataxic mouse models). While epifluorescent microscopy enables fast imaging withlarge 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 ofimplanted fiberoptic microprobes are required to ensure that voluntary and involuntaryspastic movements do not cause motion artifact in the acquired data.The Cellvizio® LAB In Vivo Confocal Fluorescence Microscope overcomes these limitationsand thereby provides an opportunity to longitudinally image the deep brain in situ withsubcellular (3.3μm) resolution, enabling unique research studies with a simultaneouscorrelation to behavioral performance. Importantly, Cellvizio LAB is the only commerciallyavailable solution for such sophisticated study.The Cellvizio LAB consists of a point-scanning confocal laser which improves image qualityby limiting out-of-focus light while still allowing a 300μm diameter field of view.Furthermore, the system is capable of 10ms frame acquisition (200 frames per second) andemploys a single-pixel avalanche photodiode detector (APD) for superior temporal resolutionand sensitivity, respectively.The recent development of the CerboFlex™ Probe and NeuroPak™ Deep Brain ImagingSystem provide a lightweight solution for chronic imaging of in situ deep brain in freelymoving mice. The CerboFlex is a fiberoptic microprobe comprised of (tens of) thousands ofindividual step-index fiber optics encased within a single bundle 300um in diameter. Non-ordered arrangement of these fibers eliminates crosstalk between adjacent fibers andmaintains high contrast and image quality. The NeuroPak System employs surgicalimplantation of a <290mg stabilization plate which allows for a sturdy, mechanical, non-permanent connection of the CerboFlex. Because the CerboFlex itself is not permanentlyimplanted, it can be removed, cleaned, and recalibrated prior to every imaging session,ensuring reliable results and quantification in longitudinal studies of the same experimentalanimal.This note explains how researchers from the Institut Pasteur (Paris, France) used theCellvizio LAB to image neurons in the Hippocampus (see supplemental data), Sn, and VTA infreely behaving mice.VisualSonics White Paper: In vivo Fiberoptic Fluorescence Microscopy in freely behaving mice Page 2
  5. 5. Experimental setupAnimalsTransgenic mice (Thy1-CerTN-L15; Heim et al., 2007) expressing GFP were used forimaging the substantia nigra while Th-gfp mice (stably transfected with a vector engineeredto express GFP under the rat tyrosine hydroxylase promoter; Sawamoto et al., 2001) wereemployed for imaging the VTA.Animals were anaesthetized by intra-peritoneal injection of Ketamine/Xylazine (0.1/0.01 mgper gram of body weight). Alternatively, animals can be anesthetized using inhaledisofluorane (3% in Oxygen) which ensures continuous immobilization and allows for fasterrecovery.Materials  NeuroPak Deep Brain Imaging SystemFigure 1. The NeuroPak Deep Brain Imaging System includes the CerboFlex Probe, 6implants and required screws, a guide holder for use with a stereotaxic device and completeprocedural information for performing the implant surgery. Shown on the right is theimplant itself, with the guide post and cannula. The implant weighs less than 300mgVisualSonics White Paper: In vivo Fiberoptic Fluorescence Microscopy in freely behaving mice Page 3
  6. 6.  CerboFlexFigure 2. The CerboFlex Probe mounted on the implant. The flexible fiber probe is shownprotruding from the cannula. The depth of penetration may be accurately controlled duringinsertion using a stereotaxic device. The fiber is 300 microns in diameter, with a beveled tipfor 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™ softwareStereotaxic coordinatesThe guide-cannula is stereotaxically inserted in the mouse brain above the targeted brainarea and the CerboFlex then lowered to the anatomical target according to bregmacoordinates (Paxinos and Franklin, The Mouse brain in stereotaxic coordinates; AcademicPress). 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).VisualSonics White Paper: In vivo Fiberoptic Fluorescence Microscopy in freely behaving mice Page 4
  7. 7. ProcedureThe experiment required two distinct phases – surgical implantation (1) and imaging (2).1) At least one week prior the first imaging session the mouse underwent stereotaxicsurgery to implant a guide-cannula into the skull above the targeted structures (Seeappendix II for supplemental information).2) For imaging, mice were anesthetized and the CerboFlex imaging microprobe wasstereotaxically guided to the target structures according to Z coordinates until fluorescentneurons are identified *. The CerboFlex was then mechanically secured to the guide-cannulausing 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 oftime thereafter while freely behaving in an open field cage. At the end of individual imagingexperiments, animals were re-anesthetized to carefully remove the CerboFlex from theguide-cannula under stereotaxic guidance.*note that calibration steps were made according to Cellvizio LAB guidelines immediatelyprior to this step.Figure 3 : Thy1-CerTN-L15 mouse with a guide-cannula implanted above the Sn.VisualSonics White Paper: In vivo Fiberoptic Fluorescence Microscopy in freely behaving mice Page 5
  8. 8. ResultsThe CerboFlex microprobe was introduced into an anesthetized Th-GFP mouse through thesurgically implanted guide-cannula and into the VTA under stereotaxic guidance accordingto Z coordinates until fluorescent neurons were identified (Figure 4a, t=0).As the animal recovered from anesthetic, spastic movements did not alter the position ofthe CerboFlex relative to the VTA as evidenced by the retention of the original imaging fieldof view acquired (Figure 4b, t=20min). Fluorescent dopaminergic neurons within the VTAwere 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 qualitydue to photobleaching did not ‘appreciably affect’ detection and visualization of fluorescentneurons 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 sequencesof images acquired at several time points. The red arrows point to a brightly fluorescentneuron in the VTA of a Th-GFP mouse. Notice that the field of view remains unchangedthroughout the experiment.VisualSonics White Paper: In vivo Fiberoptic Fluorescence Microscopy in freely behaving mice Page 6
  9. 9. Similar results have been obtained for neurons of the Substantia nigra in Thy1-CerTN-L15mice over a much longer period of time. In order to reduce photobleaching and maintainsensitivity, intermittent image acquisition with 100% laser intensity was limited to 10second sequences repeated several times. As shown in Figure 5, a brightly fluorescentneuron was observed for more than 3 hours without image distortion and with minimaldepreciation of image quality.Figure 5: Images of fluorescent neurons within the Substantia nigra of a Thy1-CerTN-L15mouse. The Substantia nigra was identified according to stereotaxic coordinates andobservable fluorescence. The field of view was stable for more than 3 hours, sufficient forsimultaneous study of cellular and organismal behavior.VisualSonics White Paper: In vivo Fiberoptic Fluorescence Microscopy in freely behaving mice Page 7
  10. 10. While these studies were carried out in transgenic animals, similar results have beenobtained in the dorsal part of the mouse hippocampus (figure 9) in normal mice afterinjection of a modified Adeno Associated Virus (AAV) vector that induces cytoplasmicexpression of GFP (See supplemental information).DiscussionOwing to the confocal approach, nonordered fiberoptic bundle, and advanced algorithms ofthe Cellvizio LAB and CerboFlex along with the lightweight, stable and semi-permanentdesign of the NeuroPak, the images of fluorescent neurons in the VTA and Sn of freelymoving mice was devoid of motion artifact.Image stability is crucial when one considers the cellular study of epileptic events in animalsduring seizure where convulsive behavior is expected. It is also important when quantifyingcellular events that occur over a longer timescale such as neuronal migration. Davenne etal.(2005) elegantly quantified the velocity of neuroblasts migrating in vivo from thesubventricular zone along the rostral migratory steam to the olfactory bulb in anesthetizedadult mice. Changes in the number, direction, and velocity of migrating cells may beactivity-dependent in response to olfactory stimuli; indeed a clear relationship has beenshown between olfactory performance and the quantity of newborn neurons in the olfactorybulb (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 non-invasive observation of the bulbular neuronal network through the nasal cavity may serve toaddress the adaptive response required for fine adjustment of olfactory ability (Vincent etal. 2006).The concept of restructuring in the adult brain is, of course, not limited to the olfactorysystem; importantly, evidence suggests that neurogenesis occurs in the adult mammalianSn (Zhao, M. et al. 2003). Monitoring of cellular responses in the Sn of unrestrained, awakeanimals is imperative for a comprehensive understanding of etiology and progression ofSchizophrenia and Parkinson’s disease. Equally important for this understanding isfunctional imaging of neuronal activity. Specific dyes (calcium or voltage sensors, such asOregon Green Bapta-1 (OGB1)) must be injected prior the imaging session. These dyesserve as fluorescent sensors of intracellular [Ca2+], undergoing conformational changes thatchange the absorption/emission spectrum of the dye when in contact with Ca2+. Thevariation 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 anesthesiato verify the precise position of the CerboFlex tip within the brain. However, due to thesmall diameter of the microprobe and therefore consequent restricted lesion of the braintissue, longitudinal imaging of the same structure of the same animal can be acquired.Since these initial experiments, researchers at Institut Pasteur have successfully acquiredimages of the same freely moving animal over several weeks (unpublished data).While image stability throughout an individual imaging session is important, so is exactrepositioning the CerboFlex for longitudinal studies. Because the position of the guidecannula is fixed relative to the anatomical structure of interest, precision when reintroducingthe CerboFlex is ensured. Furthermore, in addition to the stereotaxic Z coordinates, imagesare acquired during the positioning of the CerboFlex which thereby provides another point ofVisualSonics White Paper: In vivo Fiberoptic Fluorescence Microscopy in freely behaving mice Page 8
  11. 11. reference when fluorescence is restricted to specific brain areas.The Cellvizio LAB is a unique, commercially available imaging system that affords scientiststhe opportunity to image neurons in vivo and in situ of freely moving animals, even in thedeepest parts of the brain. Due to the small diameter of the CerboFlex tip and to theflexibility of the probe, small animals can move freely in their environment or variousbehavioral mazes while images of the neuronal network are acquired over long periods oftime in longitudinal studies of the same animal.The Cellvizio LAB In Vivo Confocal Fluorescence Microscope the CerboFlex Deep BrainImaging Probe, and the NeuroPak Deep Brain Imaging System are distributed globally byVisualSonics and its distribution partners. For more information, please visitwww.visualsonics.comVisualSonics White Paper: In vivo Fiberoptic Fluorescence Microscopy in freely behaving mice Page 9
  12. 12. References 1. Michael Eisenstein (2009) Getting inside their minds Nature Methods; 6, 773-781 2. Chang JY et al 2008 3. 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, 8354–8365 4. 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. 5. 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. 6. Gheusi, G. et al. (2000) Importance of newly generated neurons in the adult olfactory bulb for odor discrimination. PNAS 97, 1823–1828 7. Heim et al. (2007), Nature Methods, 4(2):127-129 8. Holschneider DP and Maarek JM 2008 9. Mayford M et al. 1997 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, 2679–2689 12. Sawamoto et al. (2001), PNAS, 98(11): 6423–6428 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)VisualSonics White Paper: In vivo Fiberoptic Fluorescence Microscopy in freely behaving mice Page 10
  13. 13. Supplemental InformationImaging 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 ofthe dorsal hippocampus in a wildtype mouse.Prior the fixation of the headstage on the skull and guide-cannula insertion above the dorsalhippocampus, the vector was injected into the dorsal hippocampus (0.5 μl / 5 min). A delayof 3 weeks before the imaging sessions was required to ensure adequate expression of GFPwithin 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 mouseand neurons were imaged in sequences of 10 sec length to reduce photobleaching. Imagestability was observed for more than 4 hours as shown in figure S2.VisualSonics White Paper: In vivo Fiberoptic Fluorescence Microscopy in freely behaving mice Page 11
  14. 14. VisualSonics White Paper: In vivo Fiberoptic Fluorescence Microscopy in freely behaving mice Page 12
  15. 15. Figure S2: Numerous neurons of the dentate gyrus within the dorsal hippocampus afterexpression of GFP induced by injection of a modified Adeno Associated Virus (AAV) vector.Images were acquired at different time points.VisualSonics White Paper: In vivo Fiberoptic Fluorescence Microscopy in freely behaving mice Page 13
  16. 16. Appendix I: Implantation of the guide-cannulaVisualSonics White Paper: In vivo Fiberoptic Fluorescence Microscopy in freely behaving mice Page 14
  17. 17. VisualSonics White Paper: In vivo Fiberoptic Fluorescence Microscopy in freely behaving mice Page 15
  18. 18. Appendix II: Preparation of OGB1-AMSolution preparationUse one vial of OGB 488 BAPTA-1AM (Oregon Green 488 BAPTA AM-1, MW 1258.07 g,available from Invitrogen #O-6807) 1. Add 4µl of 20% pluronic acid (Invitrogen #P-3000MP) in DMSO 2. Vortex for 3 mins 3. After this step, the color of the solution should be slightly yellow 4. Add 36µL of Ca2+-free ACSF 5. Add 1µL of SR101 (2.5mM or 2mM mixed in ACSF) 6. Vortex for about 3min 7. Sonicate on ice for 5min 8. If dye sits longer than 30min, sonicate again for 5min 9. Pipette dye into centrifuge filter (Ultrafree MC, available from Fisher Scientific; UFC30GV25) 10. Centrifuge for 30 sec 11. Dilute 1: 10 in a solution containing (in mM): 150 NaCl, 2.5 KCl, 10 Hepes, pH 7.4. 12. The final solution concentration is 1mM 13. Fill pipette with approximately 8μlPractically – add 3.97 microliters of DMSO pluronic corresponding to a 10 mM solution) in avial of OGB1 and dilute the solution in 35.7 microliters of Hepes solution to obtain a 1mMsolutionEjection ParametersInjection is done using a perfusion pump through a 36G stainless steel needle: 1microliter in10 minutes (0.1μl/min). Do not remove the needle for 10 minutes. Retraction should beincremental over a 5 minute period. Following retraction, wait one hour prior to imaging.Reference: Stosiek C et al. (2003)VisualSonics White Paper: In vivo Fiberoptic Fluorescence Microscopy in freely behaving mice Page 16

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