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1、Brain Maps on the Go: Functional Imaging During Motor Challengein AnimalsDP Holschneider1,2,3,4,5 and J-M I Maarek41Department of Psychiatry and the Behavioral Sciences, Keck School of Medicine, University of SouthernCalifornia, Los Angeles, CA, USA2Department of Neurology, Keck School of Medicine,
2、University of Southern California, Los Angeles, CA,USA3Department of Cell and Neurobiology, Keck School of Medicine, University of Southern California, LosAngeles, CA, USA4Department of Biomedical Engineering, Viterbi School of Engineering, University of Southern California,Los Angeles, CA, USA5Grea
3、ter Los Angeles VA Healthcare System, Los Angeles, CA, USAAbstractBrain mapping in the freely-moving animal is useful for studying motor circuits, not only because itavoids the potential confound of sedation or restraints, but because activated brain states may serveto accentuate differences that on
4、ly manifest partially while a subject is in the resting state. Perfusionor metabolic mapping using autoradiography allows one to examine changes in brain function at thecircuit level across the entire brain with a spatial resolution ( 100 microns) appropriate for the rator mouse brain, and a tempora
5、l resolution (seconds minutes) sufficient for capturing acute brainchanges. Here we summarize the application of these methods to the functional brain mapping ofbehaviors involving locomotion of small animals, methods for the three dimensional reconstructionof the brain from autoradiographic section
6、s, voxel based analysis of the whole brain, and generationof maps of the flattened rat cortex. Application of these methods in animal models promises utilityin improving our understanding of motor function in the normal brain, and of the effects ofneuropathology and treatment interventions such as e
7、xercise have on the reorganization of motorcircuits.KeywordsBrain Mapping; neuroplasticity; neurorehabilitation; motor activity; sport sciencesCorresponding Author: D.P. Holschneider, University of Southern California, Keck School of Medicine, Dept. of Cell and Neurobiology,1333 San Pablo St., BMT 4
8、03, MC 9112, Los Angeles, CA 90033 Telephone: 1-323-442-1585, Fax: 1-323-442-1587, E-mail:holschneusc.edu.Publishers Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customerswe are providing this early version of the manuscript. Th
9、e manuscript will undergo copyediting, typesetting, and review of the resultingproof before it is published in its final citable form. Please note that during the production process errors may be discovered which couldaffect the content, and all legal disclaimers that apply to the journal pertain.NI
10、H Public AccessAuthor ManuscriptMethods. Author manuscript; available in PMC 2009 August 1.Published in final edited form as:Methods. 2008 August ; 45(4): 255261. doi:10.1016/j.ymeth.2008.04.006.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author Manuscript名师资料总结 - - -精品资料欢迎下载 - - - - - -
11、- - - - - - - - - - - - 名师精心整理 - - - - - - - 第 1 页，共 15 页 - - - - - - - - - IntroductionFunctional brain imaging has in recent years increasingly been applied to studying brainfunction related to motor activity. Mapping neural circuits aids in understanding the roleprimary and alternate circuits pla
12、y in the performance of specific motor tasks, and hererepresents a promising approach for increasing our understanding in the fields of sportssciences and neurorehabilitative medicine. Recent attention has been drawn to the fact that thetype of task and the way it is performed has different effects
13、on the brain. For instance, Lewiset al. (1) and others (2-5) have highlighted the dominance of the cerebellar-thalamic-corticalcircuit during the performance of externally guided motor movement, and contrasted this withthe dominance of the basal ganglia-thalamic-cortical circuit during the performan
14、ce ofinternally guided motor movements. Past studies (6-8), as well as our own work (9) havehighlighted the fact, that in response to brain lesions, there is both increased reliance onremaining neurons within damaged circuits, as well as new recruitment of alternate circuits.Imaging can also provide
15、 information about neurotransmitter release within specific circuits,and has been extensively developed for the study of dopamine transmission at D2 receptors.For example, Goerendt et al (2003) have used 11C-raclopride positron emission tomography(PET) to investigate levels of striatal dopamine rele
16、ase in healthy volunteers and earlyunilateral Parkinsons Disease (PD) patients while performing simple sequential fingermovements (10).Functional brain imaging can yield biomarkers for evaluating brain changes over time, eitherin response to disease or in response to neuroactive interventions, inclu
17、ding exercise. Duringrecovery from brain damage, imaging can help evaluate the presence of diaschisis (loss offunction due to cerebral lesions in areas remote from the lesion but neuronally connected toit), functional redundancies, sensory substitution and morphological changes. In response toexerci
18、se, imaging can help in addressing basic questions such as “Does exercise training resultin a neural remapping of cerebral function, and if so, where in the brain is this occurring?” Inaddition, brain mapping may provide useful endpoints for defining parameters for exercisetraining as a treatment in
19、tervention in specific neurodegenerative (11,12) or mood disorders(13,14). Currently there is no systematic investigation on the relationship between exercise anddynamic brain activation, what parameters constitute effective training (type, duration,frequency, intensity, voluntary versus forced), an
20、d what is the persistence of any changes upondiscontinuing exercise. Future identification of brain regions demonstrating changes in neuralactivation may provide guidance for establishing specific rehabilitation strategies for patients,as well as providing the basis for studies targeting molecular m
21、echanisms.A powerful means of testing motor circuits is to examine the effects acute motor challengeshave on functional brain activation. Such activated brain states may serve to accentuatedifferences that only manifest partially while a subject is in the resting state. For instance, brainmapping pe
22、rformed during motor challenges has been performed in early PD (15-17) and inanimal models of PD (9), and here has shown utility in unmasking underlying differencesbetween normal and pathological neural circuits.Undertaking to answer these questions in an animal model, rather than in a clinical popu
23、lation,provides distinct advantages. It obviates problems related to recruitment, compliance andretention of patients. Functional neuroimaging can also be performed in the freely-movinganimal during a locomotor challenge, which presents significant technical challenges in clinicalstudies. Finally, a
24、nimal studies allow correlation between motor functional testing, functionalbrain activation and histologic outcomes.In the past, functional brain activation has been studied in nontethered, freely moving animalswith a variety of modalities. Brain electrical recordings using radiotelemetry offer the
25、Holschneider and MaarekPage 2Methods. Author manuscript; available in PMC 2009 August 1.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author Manuscript名师资料总结 - - -精品资料欢迎下载 - - - - - - - - - - - - - - - - - - 名师精心整理 - - - - - - - 第 2 页，共 15 页 - - - - - - - - - advantage of detailed informati
26、on regarding the temporal and spatial synchronization of neuralprocesses, however, only limited cortical and subcortical areas can be mappedelectrophysiologically in a single subject. Brain functional activation in animals has also beenexplored at the cellular level using measurement of changes in c
27、-fos, an early gene product(18-20), 5-3H-uridine uptake (21,22), a nucleic acid important in neuronal RNA synthesis, orcytochrome oxidase, a mitochondrial enzyme (23-26). These indirect markers of neural activityprovide excellent spatial resolution, however, the temporal resolution is very poor, req
28、uiringlong-lasting stimulation. Spatial resolution, with micro-PET and advanced imagereconstruction software, remains at best 1.6 mm at the center of the field of view. Thisrepresents 10% and 18% of the width of the rat and mouse brain, respectively and is poorlysuited for the detection for all but
29、the broadest changes in regional cerebral blood flow (CBF)or metabolism (CMR). Functional magnetic resonance imaging (fMRI) and single photonemission computed tomography (SPECT, microSPECT), though they provide whole brainanalysis and adequate spatial and temporal resolution, require sedation of the
30、 animal, limitingthe behaviors that can be examined. We have made use of perfusion or metabolic mappingusing autoradiography for the brain mapping in small animals. Autoradiographic methodsallow one to examine changes in brain function at the circuit level across the entire brain, witha spatial reso
31、lution ( 100 microns) appropriate for the rat or mouse brain, and a temporalresolution (seconds minutes) sufficient for capturing acute brain changes.To facilitate brain mapping during locomotor activity, our laboratory has developed a self-contained, fully implantable miniature infusion pump (MIP)
32、that in small animals (27,28)allows bolus injection of pharmacologic agents by remote activation. The ability to trigger thispump by remote activation has allowed us to rapidly inject cerebral blood flow tracers in thenonrestrained, nontethered animal, thereby allowing the brain mapping of behaviors
33、 involvinglocomotion. Currently, no such device exists on the market, with osmotic (Alzet, Durect Corp.,Cupertino, CA), elastomeric (VIP, Advanced Neuromodulation Systems, Plano, TX),electrolytic (Infu-Disk, Med-e-cell, San Diego, CA), peristaltic (iPRECIO, Primetech Corp.,Tokyo, Japan; Pegasus, Ins
34、tech Laboratories, Inc., Plymouth Meeting, PA) minipumpsproviding only slow infusion rates, without the ability for user-initiated remote activation. Wehave validated the MIP as a tool for functional neuroimaging in rats in which a perfusion tracersuch as 14C-iodoantipyrine is administered with the
35、animal in the freely-moving state, andbrain mapping of the regional distribution of cerebral blood flow occurs in the cryosectionedbrain using autoradiography. Generation of functional images of motor tasks (29,30), ofauditory center activation in response to an acoustic challenge (31) and limbic ar
36、eas duringconditioned fear memory (32) has provided strong evidence of the unique ability of the MIPto produce “snapshots ” of the brain functional activation during a behavior. The MIP hasprovided a new tool for functional neuroimaging, and opened opportunities for performing“brain scans on the mov
37、e” (http:/www.nibib.nih.gov/publicPage.cfm?pageid=657). To allowus to analyze and display autoradiographic data obtained with the MIP, we have developedmethods for the three dimensional reconstruction of the rat brain from autoradiographicsections, voxel based analysis of the whole brain, and genera
38、tion of perfusion maps of theflattened rat cortex. Below, we summarize methods related to use of the MIP in mappinglocomotor behavior, as well as methods for the subsequent data analysis.MethodsStandardizing complex behaviors during brain mappingAnimals of uniform age, strain and gender are chosen.
39、Because motor behaviors involvemultisensory input to the animal, standardization of not only the motor paradigm (e.g. speed,duration, etc.), but also of the experimental environment is necessary. Ambient light levels andsound levels should be kept low and standardized using a hand-held light meter (
40、Control Co.,Holschneider and MaarekPage 3Methods. Author manuscript; available in PMC 2009 August 1.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author Manuscript名师资料总结 - - -精品资料欢迎下载 - - - - - - - - - - - - - - - - - - 名师精心整理 - - - - - - - 第 3 页，共 15 页 - - - - - - - - - TX) and sound meter
41、 (Radioshak, TX). Olfactory cues are minimized by wiping theexperimental arena with water and alcohol. Time of day, duration of testing, room temperatureis standardized. No entry in and out of the experimental room by staff should be permittedduring the imaging.Care must be taken to define appropria
42、te controls such that group differences in brain activationcan be ascribed to the motor paradigm itself, and not to differences in the animals mental state(e.g. habituation, novelty, anxiety) that could be eliminated through standardization of theprotocol. To overcome the animals natural fear and an
43、xiety, rats are equally handled prior tobehavioral testing, and are familiarized with the paradigms prior to the start of the experiments.“Novelty ” effects are avoided by prior exposure to the behavioral paradigm on severaloccasions. On the day of experimentation, animals are habituated to the expe
44、rimental room for1 hour prior to testing, and then again for 45 minutes after loading of the radiotracer into theMIP. Use of detailed kinematic scoring may be useful for providing an independent measureof the uniformity of the motor challenge and in evaluating behavioral compensation strategiesthat
45、may occur during recovery from damage to the nervous system. Such measures can beintroduced as covariates in the brain mapping analysis to evaluate the role alternate behavioralstrategies may play in eliciting alternate patterns of activation. Kinematic measures can beobtained by video recordings wi
46、th a high speed video camera (1000 frames/sec) during theperiod of tracer injection (10 seconds), as well as during an earlier baseline period. Offlineanalysis of such behaviors can occur using the Observer (Noldus, Inc.), a software programthat could be used for the manual coding of behaviors, for
47、instance, footslips, dominance ofhindlimb versus forelimb, and carry of the limbs and posture of the animal. Alternatively, moredetailed kinematic analyses can be provided by programs such as Motus (Vicon, CA), Catwalkor Ethovision (Noldus, Inc., VA) depending on the application.Microbolus infusion
48、pumpDetails of the design and fabrication of the first and second generation of the MIP have beenpublished (27,33). The MIP consists of an intravenous catheter, a silicone embeddedelectronics controller remotely activated by a photodetector that responds to trains of lightpulses (30 KHz frequency) a
49、nd controls a normally-closed miniaturized solenoid valve, anejection chamber containing the radiotracer, and a silastic reservoir containing a euthanasiasolution. The photodetector with peak spectral sensitivity in the near infrared (NIR) spectrumof wavelengths allows for transcutaneous triggering
50、of the pump with trains of light pulsesfrom external NIR LEDs in the experimental room. Upon triggering, the microvalve opens,allowing the hydraulic pressure from the reservoir to release first the radiotracer into theanimals circulation, followed a few seconds thereafter by a euthanasia agent (1.0