Name: pupillometry to assess auditory sensation in guinea pigs
Uploaded: 2023-01-06T13:25:00
Description: Watch this Scientific Journal Video about Pupillometry to Assess Auditory Sensation in Guinea Pigs at JoVE.com

Dette arbejde blev støttet af NIH (R01DC017141), Pennsylvania Lions Hearing Research Foundation og midler fra afdelingerne for otolaryngologi og neurobiologi, University of Pittsburgh.

For alle eksperimentelle procedurer skal du opnå godkendelse fra Institutional Animal Care and Use Committee (IACUC) og overholde NIH-retningslinjerne for pleje og brug af forsøgsdyr. I USA er praktiserende læger desuden underlagt reglerne fra USA's landbrugsministerium (USDA). Alle procedurer i denne protokol blev godkendt af University of Pittsburgh IACUC og overholdt NIH-retningslinjerne for pleje og brug af forsøgsdyr. Til dette eksperiment blev der anvendt tre mandlige vildtype, pigmenterede læger mellem 4 og 1...

Denne protokol demonstrerer brugen af pupillometri som en ikke-invasiv og pålidelig metode til at estimere auditive tærskler hos passivt lyttende dyr. I overensstemmelse med den her beskrevne protokol blev der estimeret tærskler for kalkulering af støj hos normalthørende læger. Tærskler beregnet ved hjælp af pupillometri viste sig at være i overensstemmelse med dem, der blev opnået ved hjælp af operant træning62. Sammenlignet med operant træning var pupillometriprotokollen imidlertid ...

Pupildiameter (PD) kan påvirkes af en lang række faktorer, og måling af PD, der ændrer sig over tid, kaldes pupillometri. PD styres af irislukkemusklen (involveret i indsnævring) og irisdilatatormusklen (involveret i udvidelse). Indsnævringsmusklen er innerveret af det parasympatiske system og involverer kolinerge fremspring, mens irisdilatatoren er innerveret af det sympatiske system, der involverer noradrenerge og kolinerge fremspring 1,2,3. Den mest kendte stimulus til at inducere PD-ændringer er luminans-indsnævring og dilatationsrespons af pupillen kan produceres ved variationer i omgivende lysintensitet2. PD ændres også som funktion af brændvidde2. Det har imidlertid været kendt i årtier, at PD også viser ikke-luminansrelaterede udsving 4,5,6,7. For eksempel kan ændringer i interne mentale tilstande fremkalde forbigående PD-ændringer. Eleven udvider sig som reaktion på følelsesladede stimuli eller øges med ophidselse 4,5,8,9. Pupiludvidelse kan også være relateret til andre kognitive mekanismer, såsom øget mental indsats eller opmærksomhed10,11,12,13. På grund af dette forhold mellem pupilstørrelsesvariationer og mentale tilstande er PD-ændringer blevet undersøgt som en markør for kliniske lidelser som skizofreni 14,15, angst 16,17,18, Parkinsons sygdom 19,20 og Alzheimers sygdom 21 , blandt andre. Hos dyr sporer PD-ændringer interne adfærdstilstande og er korreleret med neuronale aktivitetsniveauer i kortikale områder22,23,24,25. Pupildiameter har også vist sig at være en pålidelig indikator for søvntilstanden hos mus26. Disse PD-ændringer relateret til ophidselse og den interne tilstand forekommer typisk på lange tidsskalaer i størrelsesordenen flere titalls sekunder.
Inden for høreforskning, ved normal hørelse såvel som hos hørehæmmede forsøgspersoner er lytteanstrengelse og auditiv opfattelse blevet vurderet ved hjælp af pupillometri. Disse undersøgelser involverer typisk uddannede forskningsemner27,28,29,30, der udfører forskellige former for detektions- eller genkendelsesopgaver. På grund af det førnævnte forhold mellem ophidselse og PD har øget opgaveengagement og lytteindsats vist sig at være korreleret med øgede pupiludvidelsesresponser 30,31,32,33,34,35. Således er pupillometri blevet brugt til at demonstrere, at der bruges øget lytteindsats til at genkende spektralt forringet tale hos normalthørende lyttere 29,36. Hos hørehæmmede lyttere, såsom mennesker med aldersrelateret høretab 27,30,37,38,39,40,41 og cochlear implantatbrugere 42,43, steg pupillens respons også med faldende taleforståelighed; Imidlertid viste hørehæmmede lyttere større pupiludvidelse under lettere lytteforhold sammenlignet med normale hørepersoner 27,30,37,38,39,40,41,42,43. Men eksperimenter, der kræver, at lytteren udfører en genkendelsesopgave, er ikke altid mulige - for eksempel hos spædbørn eller i nogle dyremodeller. Således kan ikke-luminansrelaterede pupilresponser fremkaldt af akustiske stimuli være en levedygtig alternativ metode til vurdering af auditiv detektion i disse tilfælde44,45. Tidligere undersøgelser viste en forbigående og stimulus-bundet pupiludvidelse som en del af orienteringsrefleksen46. Senere undersøgelser har vist brugen af stimulus-bundne pupildilatationer til at udlede frekvensfølsomhedskurver hos ugler47,48. For nylig er disse metoder blevet tilpasset til at vurdere følsomheden af pupildilatationsresponsen hos spædbørn48. Pupillometri har vist sig at være en pålidelig og ikke-invasiv tilgang til estimering af auditiv detektion og diskriminationstærskler hos passivt lyttende marsvin (GP'er) ved hjælp af en bred vifte af enkle (toner) og komplekse (GP-vokaliseringer) stimuli49. Disse stimulusrelaterede PD-ændringer forekommer typisk på hurtigere tidsskalaer i størrelsesordenen flere sekunder og er knyttet til stimulustiming. Her foreslås pupillometri af stimulusrelaterede PD-ændringer som en metode til at studere adfærdsmæssige virkninger af forskellige former for hørenedsættelse i dyremodeller. Især pupillometriprotokoller til brug i praktiserende læger, en veletableret dyremodel af forskellige typer auditive patologier 50,51,52,53,54,55,56 (se også reference 57 for en udtømmende gennemgang) beskrives.
Selvom denne teknik er demonstreret hos normalthørende læger, kan disse metoder let tilpasses andre dyremodeller og dyremodeller af forskellige auditive patologier. Det er vigtigt, at pupillometri kan kombineres med andre ikke-invasive målinger såsom EEG samt med invasive elektrofysiologiske optagelser for at studere mekanismerne bag mulige lyddetekterings- og opfattelsesunderskud. Endelig kan denne tilgang også bruges til at etablere brede ligheder mellem menneske- og dyremodeller.

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			<td>Tang Band Speaker, Taipei, Taiwan</td>
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			<td>Tucker-Davis Technologies, Alachua, FL</td>
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			<td>Viewpoint software</td>
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pupillometry to assess auditory sensation in guinea pigs

Støjeksponering er en førende årsag til sensorineuralt høretab. Dyremodeller for støjinduceret høretab har skabt mekanistisk indsigt i høretabets underliggende anatomiske og fysiologiske patologier. Imidlertid er det stadig udfordrende at relatere adfærdsmæssige underskud observeret hos mennesker med høretab til adfærdsmæssige underskud i dyremodeller. Her foreslås pupillometri som en metode, der muliggør direkte sammenligning af dyre- og menneskelige adfærdsdata. Metoden er baseret på et modificeret oddball-paradigme - vænner emnet til den gentagne præsentation af en stimulus og intermitterende præsenterer en afvigende stimulus, der varierer på en vis parametrisk måde fra den gentagne stimulus. Den grundlæggende forudsætning er, at hvis ændringen mellem den gentagne og afvigende stimulus detekteres af emnet, vil det udløse et pupiludvidelsesrespons, der er større end det, der fremkaldes af den gentagne stimulus. Denne tilgang demonstreres ved hjælp af en vokaliseringskategoriseringsopgave hos marsvin, en dyremodel, der i vid udstrækning anvendes i auditiv forskning, herunder i høretabsundersøgelser. Ved at præsentere vokaliseringer fra en vokaliseringskategori som standardstimuli og en anden kategori som oddball-stimuli indlejret i støj ved forskellige signal-støj-forhold, demonstreres det, at størrelsen af pupiludvidelse som reaktion på oddball-kategorien varierer monotont med signal-støj-forholdet. Vækstkurveanalyser kan derefter bruges til at karakterisere tidsforløbet og den statistiske signifikans af disse pupildilatationsresponser. I denne protokol beskrives detaljerede procedurer for akklimatisering af marsvin til opsætningen, udførelse af pupillometri og evaluering / analyse af data. Selvom denne teknik er demonstreret hos normalthørende marsvin i denne protokol, kan metoden bruges til at vurdere de sensoriske virkninger af forskellige former for høretab inden for hvert emne. Disse virkninger kan derefter korreleres med samtidige elektrofysiologiske målinger og post-hoc anatomiske observationer.

Pupillometri blev udført hos tre mandlige pigmenterede læger, der vejer ~ 600-1.000 g i løbet af forsøgene. Som beskrevet i denne protokol blev der brugt et oddball-paradigme til stimuluspræsentation for at estimere opkalds-støj-kategoriseringstærskler. I oddball-paradigmet blev kald, der tilhørte en kategori (hvin) indlejret i hvid støj ved en given SNR, anvendt som standardstimuli (figur 2A), og kald fra en anden kategori (hvæser) indlejret i hvid støj ved samme SNR (figur 2A) s...

Watch this Scientific Journal Video about Pupillometry to Assess Auditory Sensation in Guinea Pigs at JoVE.com

Pupillometry to Assess Auditory Sensation in Guinea Pigs

Pupillometri, en simpel og ikke-invasiv teknik, foreslås som en metode til bestemmelse af høretærskler i støj hos normale høredyr og dyremodeller af forskellige auditive patologier.

Pupillometri til vurdering af auditiv fornemmelse hos marsvin

Noise exposure is a leading cause of sensorineural hearing loss. Animal models of noise-induced hearing loss have generated mechanistic insight into the ...

Pupillometri kan bruges til at estimere komplekse lydgenkendelsestærskler, og kombineret med andre metoder kan bruges til at bestemme virkningerne af forskellige former for høretab på disse tærskler. Denne teknik kan bruges til at karakterisere data af samme modalitet fra mennesker og fra utrænede dyr. Det er relativt let at konfigurere og minimalt invasivt.Adfærds- og opfattelsesmål er vanskelige at opnå i dyremodeller for høretab. Denne metode er en måde at kvantificere kompleks lydgenkendelsesadfærd hos dyr. Som vi gør alle dyreadfærdseksperimenter, er tålmodighed nøglen.Tag dig tid til at akklimatisere dyret til forsøgsopsætningen. Hold eksperimentelle sessioner korte og overvåg dyret nøje. Til at begynde med skal du montere en kalibreret højttaler på den lyddæmpede kammervæg i samme højde som den position, hvor dyret skal placeres.For fri feltstimuleringslevering skal du placere dyret i kabinettet og sikre, at store kropsbevægelser ikke er mulige, og fastgør dyrets hoved til den stive ramme. Anbring en piezoelektrisk sensor under anlægget for at detektere og registrere dyrenes bevægelser. Placer derefter pupilbilledkameraet i en afstand af 25 centimeter fra motivets øje.For at sætte luftpustet op skal du bruge en holder, der er fastgjort til bordpladen, til at placere en pipettespids omkring 15 centimeter foran dyrets tryne. Tilslut et silikonerør med en diameter på ca. tre millimeter til pipettespidsen, og tilslut røret til en reguleret luftcylinder. Hold cylinderens lufttryk mellem 20 og 25 psi.Og før røret gennem en klemmeventil for at kontrollere timingen og varigheden af luftpustet ved hjælp af et computerstyret relæ. Oplys øjet med et infrarødt LED-array placeret på omkring 10 centimeter afstand. Brug hvid LED-belysning med en intensitet på omkring 2000 candela pr. kvadratmeter for at belyse det afbildede øje og bringe pupildiameteren til omkring 3,5 millimeter.Åbn elevoptagelsessoftwaren, og hent videoen af eleven ved hjælp af et kamera med en 16 millimeter linse med en rumlig opløsning på en 0,15 graders visuel vinkel og et infrarødt filter placeret i en afstand på 25 centimeter fra det afbildede øje. Sørg for, at øjet er centreret i det afbildede område. Reguler kameraets blænde og fokus samt IR-niveauet, indtil omridset af den afbildede pupil er i skarpt fokus.I elevoptagelsessoftwaren skal du definere det interesseområde, der indeholder eleven, ved at vælge et rektangulært område med musen. Brug derefter kontrolpanelet til at justere lysstyrken og kontrasten for den erhvervede video. Indstil scanningstætheden til fem, og juster tærsklen, så ellipsetilpasningen nøje svarer til pupillens kontur i videoen.Brug den neurale interfaceprocessorsoftware til at erhverve og gemme det analoge signal fra pupildiameteren eller PD-sporet, spændingssporet fra den piezoelektriske sensor, der registrerer bevægelse, stimulusleveringstiderne og leveringstiderne for luftpust. Vælg otte forskellige eksempler på marsvinvokaliseringer af samme længde fra to forskellige kategorier af vokaliseringer. For eksempel wheek opkald og whine opkald.Den ene kategori vil tjene som standard stimuli, og den anden kategori vil tjene som oddball eller afvigende stimuli. For at generere et sekund lange standard og afvigende stimuli indlejret i støj ved forskellige signal-støj-forhold eller SNR-niveauer, tilføj hvid støj af samme længde til opkaldene. Intervallet af SNR'er udtaget i dette eksperiment er mellem minus 24 og plus 40 decibel.For hver session skal du forberede en pseudorandom stimuluspræsentationssekvens, der indeholder standardstimuli større end 90% af tiden. Sørg for, at der mellem afvigende stimuli er mindst 20 til 40 forsøg med standardstimuli. Brug en fast stimulusintensitet til alle stimuluspræsentationer.Præsenter stimuli med høj tidsmæssig regelmæssighed. For at opretholde dyrets engagement med stimuli og for at minimere tilvænning, skal du eventuelt levere et kort luftpust efter den afvigende stimulus. Sørg for, at begyndelsen af luftpustet er tilstrækkeligt adskilt fra stimulusvarigheden, så stimulus fremkaldte pupiludvidelsesresponser.Nå et højdepunkt før luftpustinducerede blinkartefakter. Kør koden pupil_avg_JOVE_m, og vælg datafilen fra en enkelt session i pop op-dialogboksen for at udføre bevægelsesregistrering og udelukkelse af forsøg for hver session. Kør derefter den samme kode, og vælg alle de datafiler, der skal analyseres i pop op-dialogboksen.For at fjerne øjenblinkartefakter skal du forbehandle dataene og opnå den gennemsnitlige pupiludvidelse til hver stimulus på tværs af sessioner. Gennemsnit de stimulusfremkaldte pupildiameterændringer for hver stimulustilstand på tværs af sessioner inden for hvert dyr og derefter på tværs af dyr for at generere det gennemsnitlige pupiludvidelsesrespons på hver stimulustilstand. Sammenkæd alle output fra koden pupil_avg_JOVE lodret.m eller alle sessionsdyr, SNR'er og dæmpninger for at konstruere en matrix, der indeholder kolonnerne, dyre-ID, SNR, lydniveau og pupildiameterværdier. For hver SNR plottes vægtene svarende til skæringspunktet for at visualisere resultaterne. Gør det samme for lineære og kvadratiske udtryk.Sæt hele den sessionsvise procentdel af forsøg med signifikante pupilændringer i hver celle i et cellearray, hvor cellerne er arrangeret fra lavere til højere SNR. Brug koden pupil_threshold_estimate_JOVE_m til at estimere opkaldet i støjkategoriseringstærsklen. Gennemsnitlige elevsvar fra tre dyr er vist i denne figur.Gennemsnitlige pupilresponser på standard whine stimuli er repræsenteret af den blå linje, og skygge svarer til plus minus en standard fejl af middelværdi. Grå linjer og skygge svarer til middelværdi og plus minus en standardfejl af middelværdien af elevresponser fremkaldt af afvigende hvæsenstimuli. Grå skyggeintensitet svarer til SNR.Grøn linje og skygge svarer til det gennemsnitlige elevspor ved tærskel SNR. Den røde lodrette linje svarer til stimulusdebut. Den orange lodrette linje svarer til luftpustens begyndelse, og de blågrønne stiplede linjer svarer til GCA-vinduet.Den psykometriske funktion, der passer til procentdelen af forsøg med signifikante pupildiameterændringer fremkaldt af den afvigende stimulus som funktion af SNR, er vist i denne figur. Whiskers svarer til plus minus en standard fejl af middelværdi. Bemærk, at 50% af maksimum nås ved ca. minus 20 decibel SNR.De erhvervede data af høj kvalitet er det vigtigt at akklimatisere dyret godt til forsøgsopsætningen og opretholde konstante elimineringsbetingelser. Hold eksperimentsessionen kort for at minimere dyrets beboelse til de afvigende stimuli. EEG eller elektrofysiologiske optagelser kombineret med pupillometri ville generere yderligere indsigt i de neurale underskud, underliggende call-in-noise kategoriseringsunderskud hos hørehæmmede dyr.

pupillometry-to-assess-auditory-sensation-in-guinea-pigs

Research

JoVE Journal

Neuroscience

Operant Sensation Seeking in the Mouse

Operant methods are powerful behavioral tools for the study of motivated behavior. These 'self-administration' methods have been used extensively in drug addiction research due to their high construct validity. Operant studies provide researchers a tool for preclinical investigation of several aspects of the addiction process. For example, mechanisms of acute reinforcement (both drug and non-drug) can be tested using pharmacological or genetic tools to determine the ability of a molecular target to influence self-administration behavior1-6. Additionally, drug or food seeking behaviors can be studied in the absence of the primary reinforcer, and the ability of pharmacological compounds to disrupt this process is a preclinical model for discovery of molecular targets and compounds that may be useful for the treatment of addiction3,7-9. One problem with performing intravenous drug self-administration studies in the mouse is the technical difficulty of maintaining catheter patency. Attrition rates in these experiments are high and can reach 40% or higher10-15. Another general problem with drug self-administration is discerning which pharmacologically-induced effects of the reinforcer produce specific behaviors. For example, measurement of the reinforcing and neurological effects of psychostimulants can be confounded by their psychomotor effects. Operant methods using food reinforcement can avoid these pitfalls, although their utility in studying drug addiction is limited by the fact that some manipulations that alter drug self-administration have a minimal impact on food self-administration. For example, mesolimbic dopamine lesion or knockout of the D1 dopamine receptor reduce cocaine self-administration without having a significant impact on food self-administration 12,16. 
Sensory stimuli have been described for their ability to support operant responding as primary reinforcers (i.e. not conditioned reinforcers)17-22. Auditory and visual stimuli are self-administered by several species18,21,23, although surprisingly little is known about the neural mechanisms underlying this reinforcement. The operant sensation seeking (OSS) model is a robust model for obtaining sensory self-administration in the mouse, allowing the study of neural mechanisms important in sensory reinforcement24. An additional advantage of OSS is the ability to screen mutant mice for differences in operant behavior that may be relevant to addiction. We have reported that dopamine D1 receptor knockout mice, previously shown to be deficient in psychostimulant self-administration, also fail to acquire OSS24. This is a unique finding in that these mice are capable of learning an operant task when food is used as a reinforcer. While operant studies using food reinforcement can be useful in the study of general motivated behavior and the mechanisms underlying food reinforcement, as mentioned above, these studies are limited in their application to studying molecular mechanisms of drug addiction. Thus, there may be similar neural substrates mediating sensory and psychostimulant reinforcement that are distinct from food reinforcement, which would make OSS a particularly attractive model for the study of drug addiction processes. The degree of overlap between other molecular targets of OSS and drug reinforcers is unclear, but is a topic that we are currently pursuing. While some aspects of addiction such as resistance to extinction may be observed with OSS, we have found that escalation 25 is not observed in this model24. Interestingly, escalation of intake and some other aspects of addiction are observed with self-administration of sucrose26. Thus, when non-drug operant procedures are desired to study addiction-related processes, food or sensory reinforcers can be chosen to best fit the particular question being asked. 
In conclusion, both food self-administration and OSS in the mouse have the advantage of not requiring an intravenous catheter, which allows a higher throughput means to study the effects of pharmacological or genetic manipulation of neural targets involved in motivation. While operant testing using food as a reinforcer is particularly useful in the study of the regulation of food intake, OSS is particularly apt for studying reinforcement mechanisms of sensory stimuli and may have broad applicability to novelty seeking and addiction.

In this protocol we describe a method of operant learning using sensory stimuli as a reinforcer in the mouse. It requires no prior training or food restriction, and it allows the study of motivated behavior without the use of a pharmacological or natural reinforcer such as food.

Operant methods are powerful behavioral tools for the study of motivated behavior.  These 'self-administration' methods have been used extensively in drug ...

Preparation and Culture of Chicken Auditory Brainstem Slices

The chicken auditory brainstem is a well-established model system that has been widely used to study the anatomy and physiology of auditory processing at discreet periods of development 1-4 as well as mechanisms for temporal coding in the central nervous system 5-7.
Here we present a method to prepare chicken auditory brainstem slices that can be used for acute experimental procedures or to culture organotypic slices for long-term experimental manipulations.
The chicken auditory brainstem is composed of nucleus angularis, magnocellularis, laminaris and superior olive. These nuclei are responsible for binaural sound processing and single coronal slice preparations preserve the entire circuitry. Ultimately, organotypic slice cultures can provide the opportunity to manipulate several developmental parameters such as synaptic activity, expression of pre and postsynaptic components, expression of aspects controlling excitability and differential gene expression
This approach can be used to broaden general knowledge about neural circuit development, refinement and maturation.

The chicken auditory brainstem is comprised of nuclei responsible for binaural sound processing. A single coronal slice preparation maintains the entire circuitry while the cultured approach provides a unique preparation to study the development of neuronal structure and auditory function at the molecular, cellular and network levels.

The chicken auditory brainstem is a well-established model system that has been widely used to study the anatomy and physiology of auditory processing at ...

A Lightweight, Headphones-based System for Manipulating Auditory Feedback in Songbirds

Experimental manipulations of sensory feedback during complex behavior have provided valuable insights into the computations underlying motor control and sensorimotor plasticity1. Consistent sensory perturbations result in compensatory changes in motor output, reflecting changes in feedforward motor control that reduce the experienced feedback error. By quantifying how different sensory feedback errors affect human behavior, prior studies have explored how visual signals are used to recalibrate arm movements2,3 and auditory feedback is used to modify speech production4-7. The strength of this approach rests on the ability to mimic naturalistic errors in behavior, allowing the experimenter to observe how experienced errors in production are used to recalibrate motor output.
Songbirds provide an excellent animal model for investigating the neural basis of sensorimotor control and plasticity8,9. The songbird brain provides a well-defined circuit in which the areas necessary for song learning are spatially separated from those required for song production, and neural recording and lesion studies have made significant advances in understanding how different brain areas contribute to vocal behavior9-12. However, the lack of a naturalistic error-correction paradigm - in which a known acoustic parameter is perturbed by the experimenter and then corrected by the songbird - has made it difficult to understand the computations underlying vocal learning or how different elements of the neural circuit contribute to the correction of vocal errors13.
The technique described here gives the experimenter precise control over auditory feedback errors in singing birds, allowing the introduction of arbitrary sensory errors that can be used to drive vocal learning. Online sound-processing equipment is used to introduce a known perturbation to the acoustics of song, and a miniaturized headphones apparatus is used to replace a songbird's natural auditory feedback with the perturbed signal in real time. We have used this paradigm to perturb the fundamental frequency (pitch) of auditory feedback in adult songbirds, providing the first demonstration that adult birds maintain vocal performance using error correction14. The present protocol can be used to implement a wide range of sensory feedback perturbations (including but not limited to pitch shifts) to investigate the computational and neurophysiological basis of vocal learning.

We describe the design and assembly of miniaturized headphones suitable for replacing a songbird&#x2019;s natural auditory feedback with a manipulated acoustic signal. Online sound processing hardware is used to manipulate song output, introduce real-time errors in auditory feedback via the headphones, and drive vocal motor learning.

Et let, Hovedtelefoner-baseret system til at manipulere Auditiv Feedback i Songbirds

Experimental manipulations of sensory feedback during complex behavior have provided valuable insights into the computations underlying motor control and ...

Selective Tracing of Auditory Fibers in the Avian Embryonic Vestibulocochlear Nerve

The embryonic chick is a widely used model for the study of peripheral and central ganglion cell projections. In the auditory system, selective labeling of auditory axons within the VIIIth cranial nerve would enhance the study of central auditory circuit development. This approach is challenging because multiple sensory organs of the inner ear contribute to the VIIIth nerve 1. Moreover, markers that reliably distinguish auditory versus vestibular groups of axons within the avian VIIIth nerve have yet to be identified. Auditory and vestibular pathways cannot be distinguished functionally in early embryos, as sensory-evoked responses are not present before the circuits are formed. Centrally projecting VIIIth nerve axons have been traced in some studies, but auditory axon labeling was accompanied by labeling from other VIIIth nerve components 2,3. Here, we describe a method for anterograde tracing from the acoustic ganglion to selectively label auditory axons within the developing VIIIth nerve. First, after partial dissection of the anterior cephalic region of an 8-day chick embryo immersed in oxygenated artificial cerebrospinal fluid, the cochlear duct is identified by anatomical landmarks. Next, a fine pulled glass micropipette is positioned to inject a small amount of rhodamine dextran amine into the duct and adjacent deep region where the acoustic ganglion cells are located. Within thirty minutes following the injection, auditory axons are traced centrally into the hindbrain and can later be visualized following histologic preparation. This method provides a useful tool for developmental studies of peripheral to central auditory circuit formation.

Here we describe a microdissection technique followed by fluorescent dye injection into the acoustic ganglion of early chick embryos for selective tracing of auditory axon fibers in the nerve and hindbrain.

Selektiv Tracing af Auditive fibre i de Avian embryoer Vestibulocochlear Nerve

The embryonic chick is a widely used model for the study of peripheral and central ganglion cell projections. In the auditory system, selective labeling ...

Functional Imaging of Auditory Cortex in Adult Cats using High-field fMRI

Current knowledge of sensory processing in the mammalian auditory system is mainly derived from electrophysiological studies in a variety of animal models, including monkeys, ferrets, bats, rodents, and cats. In order to draw suitable parallels between human and animal models of auditory function, it is important to establish a bridge between human functional imaging studies and animal electrophysiological studies. Functional magnetic resonance imaging (fMRI) is an established, minimally invasive method of measuring broad patterns of hemodynamic activity across different regions of the cerebral cortex. This technique is widely used to probe sensory function in the human brain, is a useful tool in linking studies of auditory processing in both humans and animals and has been successfully used to investigate auditory function in monkeys and rodents. The following protocol describes an experimental procedure for investigating auditory function in anesthetized adult cats by measuring stimulus-evoked hemodynamic changes in auditory cortex using fMRI. This method facilitates comparison of the hemodynamic responses across different models of auditory function thus leading to a better understanding of species-independent features of the mammalian auditory cortex.

Functional studies of the auditory system in mammals have traditionally been conducted using spatially-focused techniques such as electrophysiological recordings. The following protocol describes a method of visualizing large-scale patterns of evoked hemodynamic activity in the cat auditory cortex using functional magnetic resonance imaging.

Funktionel Imaging af Auditory Cortex i voksne katte ved hjælp af High-felt fMRI

Current knowledge of sensory processing in the mammalian auditory system is mainly derived from electrophysiological studies in a variety of animal ...

Optogenetic Stimulation of the Auditory Nerve

Direct electrical stimulation of spiral ganglion neurons (SGNs) by cochlear implants (CIs) enables open speech comprehension in the majority of implanted deaf subjects1-6. Nonetheless, sound coding with current CIs has poor frequency and intensity resolution due to broad current spread from each electrode contact activating a large number of SGNs along the tonotopic axis of the cochlea7-9. Optical stimulation is proposed as an alternative to electrical stimulation that promises spatially more confined activation of SGNs and, hence, higher frequency resolution of coding. In recent years, direct infrared illumination of&#160;the cochlea has been used to evoke responses in the auditory nerve10. Nevertheless it requires higher energies than electrical stimulation10,11 and uncertainty remains as to the underlying mechanism12. Here we describe a method based on optogenetics to stimulate SGNs with low intensity blue light, using transgenic mice with neuronal expression of channelrhodopsin 2 (ChR2)13 or virus-mediated expression of the ChR2-variant CatCh14. We used micro-light emitting diodes (&#181;LEDs) and fiber-coupled lasers to stimulate ChR2-expressing SGNs through a small artificial opening (cochleostomy) or the round window. We assayed the responses by scalp recordings of light-evoked potentials (optogenetic auditory brainstem response: oABR) or by microelectrode recordings from the auditory pathway and compared them with acoustic and electrical stimulation.

Cochlear implants (CIs) enable hearing by direct electrical stimulation of the auditory nerve. However, poor frequency and intensity resolution limits the quality of hearing with CIs. Here we describe optogenetic stimulation of the auditory nerve in mice as an alternative strategy for auditory research and developing future CIs.

Optogenetic Stimulering af hørenerven

Direct electrical stimulation of spiral ganglion neurons (SGNs) by cochlear implants (CIs) enables open speech comprehension in the majority of implanted ...

The Use of Trace Eyeblink Classical Conditioning to Assess Hippocampal Dysfunction in a Rat Model of Fetal Alcohol Spectrum Disorders

Neonatal rats were administered a relatively high concentration of ethyl alcohol (11.9% v/v) during postnatal days 4-9, a time when the fetal brain undergoes rapid organizational change and is similar to accelerated brain changes that occur during the third trimester in humans. This model of fetal alcohol spectrum disorders (FASDs) produces severe brain damage, mimicking the amount and pattern of binge-drinking that occurs in some pregnant alcoholic mothers. We describe the use of trace eyeblink classical conditioning (ECC), a higher-order variant of associative learning, to assess long-term hippocampal dysfunction that is typically seen in alcohol-exposed adult offspring. At 90 days of age, rodents were surgically prepared with recording and stimulating electrodes, which measured electromyographic (EMG) blink activity from the left eyelid muscle and delivered mild shock posterior to the left eye, respectively. After a 5 day recovery period, they underwent 6 sessions of trace ECC to determine associative learning differences between alcohol-exposed and control rats. Trace ECC is one of many possible ECC procedures that can be easily modified using the same equipment and software, so that different neural systems can be assessed. ECC procedures in general, can be used as diagnostic tools for detecting neural pathology in different brain systems and different conditions that insult the brain.

Trace eyeblink classical conditioning (ECC) was used to assess hippocampal-dependent associative learning in adult rats that were administered a high concentration (11.9% v/v) of alcohol during early neonatal brain development. In general, ECC procedures are sound diagnostic tools for detecting brain dysfunction across many psychological and biomedical settings.

Brugen af Trace Eyeblink klassisk konditionering at vurdere hippocampus dysfunktion i en rotte Model af føtale alkohol spektrum forstyrrelser

Neonatal rats were administered a relatively high concentration of ethyl alcohol (11.9% v/v) during postnatal days 4-9, a time when the fetal brain ...

A Preclinical Model to Assess Brain Recovery After Acute Stroke in Rats

The purpose of this study was to establish and validate an animal brain ischemia model in the recovery and sequela stages. A middle cerebral artery occlusion/reperfusion (MCAO/R) model in male Sprague-Dawley rats was chosen. By changing the rat&#39;s weight (260&#8722;330 g), the thread bolt type (2636/2838/3040/3043) and the brain infarct time (2-3 h), a higher Longa&#39;s score, a larger infarct volume and a greater model success ratio were screened using the Longa&#39;s score and TTC staining. The optimum model condition (300 g, 3040 thread bolt, 3 h brain infarct time) was acquired and used in a 1-90 day observation period after reperfusion via assessment of sensorimotor functions and infarct volume. At these conditions, the bilateral asymmetry test had a significant difference from 1 to 90 days, and the grid-walking test had a significant difference from 1 to 60 days; both differences could be a suitable sensorimotor functional test. Thus, the most appropriate condition of a novel rat model in the recovery and sequela stages of brain ischemia was found: 300 g rats that underwent MCAO with a 3040 thread bolt for a 3 h brain infarct and then reperfused. The appropriate sensorimotor functional tests were a bilateral asymmetry test and a grid-walking test.

The&#160;purpose of this study is to establish and validate an animal model for research in the recovery and sequela stages of brain ischemia by testing brain infarction and sensorimotor function after middle cerebral artery occlusion/reperfusion (MCAO/R) after 1-90 days in rats.

En præklinisk model til vurdering af hjerne bedring efter akut slagtilfælde hos rotter

The purpose of this study was to establish and validate an animal brain ischemia model in the recovery and sequela stages. A middle cerebral artery ...

A Revised Surgical Approach to Induce Endolymphatic Hydrops in the Guinea Pig

Endolymphatic hydrops is an enlargement of scala media that is most often associated with Meniere&#39;s disease, though the pathophysiologic mechanism(s) remain unclear. In order to adequately study the attributes of endolymphatic hydrops, such as the origins of low-frequency hearing loss, a reliable model is needed. The guinea pig is a&#160;good model because it hears in the low-frequency regions that are putatively affected by endolymphatic hydrops. Previous research has demonstrated that endolymphatic hydrops can be induced surgically via intradural or extradural approaches that involve drilling on the endolymphatic duct and sac. However, whether it was possible to create an endolymphatic hydrops model using an extradural approach that avoided dangerous drilling on the endolymphatic duct and sac was unknown. The objective of this study was to demonstrate a revised extradural approach to induce experimental endolymphatic hydrops at 30 days post-operatively by obliterating the endolymphatic sac and injuring the endolymphatic duct with a fine pick. The sample size consisted of seven guinea pigs. Functional measurements of hearing were made and temporal bones were subsequently harvested for histologic analysis. The approach had a success rate of 86% in achieving endolymphatic hydrops. The risk of cerebrospinal fluid leak was minimal. No perioperative deaths or injuries to the posterior semicircular canal occurred in the sample. The presented method demonstrates a safe and reliable way to induce endolymphatic hydrops at a relatively quick time point of 30 days. The clinical implications are that the presented method provides a reliable model to further explore the origins of low-frequency hearing loss that can be associated endolymphatic hydrops.

This article demonstrates an extradural approach to obliterate the guinea pig endolymphatic sac and injure the endolymphatic duct with a fine pick in order to induce experimental endolymphatic hydrops.

En revideret kirurgisk tilgang til at fremkalde endolymphatic hydrops i MarsVin

Endolymphatic hydrops is an enlargement of scala media that is most often associated with Meniere&#39;s disease, though the pathophysiologic mechanism(s) ...

Direct Cannula Implantation in the Cisterna Magna of Pigs

The glymphatic system is a waste clearance system in the brain that relies on the flow of cerebrospinal fluid (CSF) in astrocyte-bound perivascular spaces and has been implicated in the clearance of neurotoxic peptides such as amyloid-beta. Impaired glymphatic function exacerbates disease pathology in animal models of neurodegenerative diseases, such as Alzheimer's, which highlights the importance of understanding this clearance system. The glymphatic system is often studied by cisterna magna cannulations (CMc), where tracers are delivered directly into the cerebrospinal fluid (CSF). Most studies, however, have been carried out in rodents. Here, we demonstrate an adaptation of the CMc technique in pigs. Using CMc in pigs, the glymphatic system can be studied at a high optical resolution in gyrencephalic brains and in doing so bridges the knowledge gap between rodent and human glymphatics.

This article presents a step-by-step protocol for the direct cannula implantation in the cisterna magna of pigs.

Direkte kanyle implantation i Cisterna Magna af svin

The glymphatic system is a waste clearance system in the brain that relies on the flow of cerebrospinal fluid (CSF) in astrocyte-bound perivascular spaces ...