Name: data acquisition and analysis in brainstem evoked response audiometry in mice
Uploaded: 2019-05-10T14:00:00
Description: Watch this Scientific Journal Video about Data Acquisition and Analysis In Brainstem Evoked Response Audiometry In Mice at JoVE.com

The authors would like to thank Dr. Christina Kolb (German Center for Neurodegenerative Diseases [DZNE]) and Dr. Robert Stark (DZNE) for their assistance in the animal breeding and animal health care. This work was financially supported by the Federal Institute for Drugs and Medical Devices (Bundesinstitut f&#252;r Arzneimittel und Medizinprodukte, BfArM, Bonn, Germany).

All animal procedures were performed according to the guidelines of the German Council on Animal Care and all protocols were approved by the local institutional and national committee on animal care (Landesamt f&#252;r Natur, Umwelt, und Verbraucherschutz, State Office of North Rhine-Westphalia, Department of Nature, Environment and Consumerism [LANUV NRW], Germany). The authors further certify that all animal experimentation was carried out in accordance with the National Institutes of Health Guide for the Care and Use ...

This protocol provides a detailed and integrative description of how to record auditory evoked brainstem responses in mice. It puts specific focus on animal pretreatment, anesthesia, and potential methodological confounding factors. The latter include, among others, gender, mouse line, age, and housing conditions. It should be noted that all these factors can have an impact on sensorineural hearing loss and fundamental aspects of auditory information processing. Thus, appropriate stratification of auditory profiling stud...

A central aspect of brain physiology is its capability to process environmental information resulting in different intrinsic or extrinsic output, such as learning, memory, emotional reactions, or motoric responses. Various experimental and diagnostic approaches can be used to characterize the electrophysiological responsiveness of individual neuronal cell types or clusters/ensembles of neurons within a stimulus-related neuronal circuitry. These electrophysiological techniques cover different spatiotemporal dimensions on the micro-, meso- and macroscale1. The microscale level includes voltage and current clamp approaches in different patch-clamp modes using, for instance, cultured or acutely dissociated neurons1. These in vitro techniques allow for the characterization of individual current entities and their pharmacological modulation2,3. An essential drawback, however, is the lack of systemic information as regards micro- and macrocircuitry information integration and processing. This impairment is partially overcome by in vitro techniques of the mesoscale, such as multielectrode arrays which allow for simultaneous extracellular multielectrode recordings not only in cultured neurons but also in acute brain slices4,5. Whereas microcircuitries can be preserved in the brain slices to a specific extent (e.g., in the hippocampus), long-range interconnections are typically lost6. Ultimately, to study the functional interconnections within neuronal circuitries, systemic in vivo electrophysiological techniques on the macroscale are the method of choice7. These approaches include, among other things, surface (epidural) and deep (intracerebral) EEG recordings which are carried out in both humans and animal models1. EEG signals are predominantly based on the synchronized synaptic input on pyramidal neurons in different cortical layers that can be inhibitory or excitatory in principal, despite the general predominance of excitatory input8. Upon synchronization, excitatory postsynaptic potential-based shifts in extracellular electrical fields are summed to form a signal of sufficient strength to be recorded on the scalp using surface electrodes. Notably, a detectable scalp recording from an individual electrode requires the activity of ten thousand of pyramidal neurons and a complex armamentarium of technical devices and processing tools, including an amplifier, filtering processes (low-pass filter, high-pass filter, notch filter), and electrodes with specific conductor properties.
In most experimental animal species (i.e., mice and rats), the human-based scalp EEG approach is technically not applicable, as the signal generated by the underlying cortex is too weak due to the limited number of synchronized pyramidal neurons9,10,11. In rodents, surface (scalp) electrodes or subdermal electrodes are thus severely contaminated by electrocardiogram and predominately electromyogram artifacts that make high-quality EEG recordings impossible9,11,12. When using unanesthetized freely moving mice and rats, it is therefore mandatory to directly record either from the cortex via epidural electrodes or from the deep, intracerebral structures to ensure the direct physical connection of the sensing tip of the lead/implanted electrode to the signal-generating neuronal cell clusters. These EEG approaches can be carried out either in a restraining tethered system setup or using the nonrestraining implantable EEG radio telemetry approach9,10,11. Both techniques have their pros and cons and can be a valuable approach in the qualitative and quantitative characterization of seizure susceptibility/seizure activity, circadian rhythmicity, sleep architecture, oscillatory activity, and synchronization, including time-frequency analysis, source analysis, etc.9,10,13,14,15,16,17.
Whereas tethered systems and radio telemetry allow for EEG recordings under restraining/semirestraining or nonrestraining conditions, respectively, related experimental conditions do not match the requirements for ABR recordings. The latter demand for defined acoustic stimuli which are presented repetitively over time with defined positions of a loudspeaker and experimental animal and controlled sound pressure levels (SPLs). This can be achieved either by head fixation under restraining conditions or following anesthesia18,19. To reduce the experimental stress, animals are normally anesthetized during ABR experimentation, but it should be considered that anesthesia can interfere with ABRs19,20.
As a general characteristic, the EEG is built up of different frequencies in a voltage range of 50-100 &#181;V. Background frequencies and amplitudes strongly depend on the physiological state of the experimental animal. In the awake state, beta (&#946;) and gamma (&#947;) frequencies with lower amplitude predominate. When animals become drowsy or fall asleep, alpha (&#945;), theta (&#952;), and delta (&#948;) frequencies arise, exhibiting increased EEG amplitude21. Once a sensory channel (e.g., the acoustic pathway) is stimulated, information propagation is mediated via neuronal activity through the peripheral and central nervous system. Such sensory (e.g., acoustic) stimulation triggers so-called EPs or evoked responses. Notably, event-related potentials (ERPs) are much lower in amplitude than the EEG (i.e., a few microvolts only). Thus, any individual ERP based on a single stimulus would be lost against the higher-amplitude EEG background. Therefore, a recording of an ERP requires the repetitive application of identical stimuli (e.g., clicks in ABR recordings) and subsequent averaging to eliminate any EEG background activity and artifacts. If ABR recordings are done in anesthetized animals, it is easy to use subdermal electrodes here.
Principally, AEPs include short-latency EPs, which are normally related to ABRs or BERA, and further, later-onset potentials such as midlatency EPs (midlatency responses [MLR]) and long-latency EPs22. Importantly, disturbance in the information processing of the auditory information is often a central feature of neuropsychiatric diseases (demyelinating diseases, schizophrenia, etc.) and associated with AEP alterations23,24,25. Whereas behavioral investigations are only capable of revealing functional impairment, AEP studies allow for precise spatiotemporal analysis of auditory dysfunction related to specific neuroanatomical structures26.
ABRs as early, short-latency acoustically EPs are normally detected upon moderate to high-intense click application, and there may occur up to seven ABR peaks (WI-WVII). The most important waves (WI-WV) are related to the following neuroanatomical structures: WI to the auditory nerve (distal portion, within the inner ear); WII to the cochlear nucleus (proximal portion of the auditory nerve, brainstem termination); WIII to the superior olivary complex (SOC); WIV to the lateral lemniscus (LL); WV to the termination of the lateral lemniscus (LL) within the inferior colliculus (IC) on the contralateral side27 (Supplementary Figure 1). It should be noted that WII-WV are likely to have more than one anatomical structure of the ascending auditory pathway contributing to them. Notably, the exact correlation of peaks and underlying structures of the auditory tract is still not fully clarified.
In audiology, ABRs can be used as a screening and diagnostic tool and for surgical monitoring28,29. It is most important for the identification of dysacusis, hypacusis, and anacusis (e.g., in age-related hearing loss, noise-induced hearing loss, metabolic and congenital hearing loss, and asymmetric hearing loss and hearing deficits due to deformities or malformations, injuries, and neoplasms)28. ABRs are also relevant as a screening test for hyperactive, intellectually impaired children or for other children who would not be able to respond to conventional audiometry (e.g., in neurological/psychiatric diseases such as ADHD, MS, autism etc.29,30) and in the development and surgical fitting of cochlear implants28. Finally, ABRs can provide valuable insight into the potential ototoxic side-effects of neuropsychopharmaceuticals, such as antiepileptics31,32.
The value of the translation of neurophysiological knowledge obtained from pharmacological or transgenic mouse models to humans has been demonstrated in numerous settings, particularly on the level of ERPs in auditory paradigms in mice and rats33,34,35. New insight into altered early AEPs and associated changes in auditory information processing in mice and rats can thus be translated to humans and is of central importance in the characterization and endophenotyping of auditory, neurological, and neuropsychiatric diseases in the future. Here we provide a detailed description of how ABRs can be successfully recorded and analyzed in mice for basic scientific, toxicological, and pharmacological purposes.

<table>
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			<td>AEP/OAE Software for RZ6 (BioSigRZ software)</td>
			<td>Tucker-Davis Technologies (TDT)</td>
			<td>BioSigRZ</td>
			<td></td>
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			<td>Binocular surgical magnification microscope</td>
			<td>Zeiss Stemi 2000</td>
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			<td>Techniplast</td>
			<td>1264C, 1290D</td>
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			<td>Carprox vet, 50mg/ml</td>
			<td>Virbac Tierarzneimittel GmbH</td>
			<td>PZN 11149509</td>
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			<td>Cold light source</td>
			<td>Schott KL2500 LCD</td>
			<td>9.705 202</td>
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			<td>Cotton tip applicators (sterile)</td>
			<td>Carl Roth</td>
			<td>EH12.1</td>
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			<td>Custom made meshed metal Faraday cage (stainless steel, 2 mm thickness, 1 cm mesh size)</td>
			<td>custom made</td>
			<td>custom made</td>
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			<td>5% Dexpanthenole (Bepanthen eye and nose creme)</td>
			<td>Bayer Vital GmbH</td>
			<td>PZN: 01578681</td>
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			<td>Disposable Subdermal stainless steel Needle 
			electrodes, 27GA, 12mm</td>
			<td>Rochester Electro-Medical, Inc.</td>
			<td>S03366-18</td>
			<td></td>
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			<td>Surgical drape sheets (sterile)</td>
			<td>Hartmann</td>
			<td>PZN 0366787</td>
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			<td>Ethanol, 70%</td>
			<td>Carl Roth</td>
			<td>9065.5</td>
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			<td>1/4&#39;&#39; Free Field Measure Calibration Mic Kit</td>
			<td>Tucker-Davis Technologies (TDT)</td>
			<td>PCB-378C0</td>
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			<td>Gloves (sterile)</td>
			<td>Unigloves</td>
			<td>1570</td>
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			<td>Graefe Forceps-curved, serrated</td>
			<td>FST</td>
			<td>11052-10</td>
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			<td>GraphPad Prism 6 Software, V6.07</td>
			<td>GraphPad Prism Software, Inc.</td>
			<td>https://www.graphpad.com/</td>
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			<td>Heat-based surgical instrument sterilizer</td>
			<td>FST</td>
			<td>18000-50</td>
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			<td>Homeothermic 
			heating blanked</td>
			<td>ThermoLux</td>
			<td>461265 / -67</td>
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			<td>Ketanest S (Ketamine), 25mg/ml</td>
			<td>Pfizer</td>
			<td>PZN 08707288</td>
			<td></td>
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		<tr>
			<td>Ringer&#8217;s solution (sterile)</td>
			<td>B.Braun</td>
			<td>PZN 01471434</td>
			<td></td>
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		<tr>
			<td>Matlab software</td>
			<td>MathWorks, Inc.</td>
			<td>https://de.mathworks.com/products/matlab.html</td>
			<td></td>
		</tr>
		<tr>
			<td>Medusa 4-Channel Low Imped. Headstage</td>
			<td>Tucker-Davis Technologies (TDT)</td>
			<td>RA4LI</td>
			<td></td>
		</tr>
		<tr>
			<td>Medusa 4-Channel Pre-Amp/Digitizer</td>
			<td>Tucker-Davis Technologies (TDT)</td>
			<td>RA4PA</td>
			<td></td>
		</tr>
		<tr>
			<td>Microphone</td>
			<td>PCB Pieztronics</td>
			<td>378C01</td>
			<td></td>
		</tr>
		<tr>
			<td>Multi Field Speaker- Stereo</td>
			<td>Tucker-Davis Technologies (TDT)</td>
			<td>MF1-S</td>
			<td></td>
		</tr>
		<tr>
			<td>Oscilloscope</td>
			<td>Tektronix</td>
			<td>DPO3012</td>
			<td></td>
		</tr>
		<tr>
			<td>Optical PC1 express card for Optibit Interface)</td>
			<td>Tucker-Davis Systems (TDT)</td>
			<td>PO5e</td>
			<td></td>
		</tr>
		<tr>
			<td>Askina Braucel pads (cellulose absorbet pads)</td>
			<td>B.Braun</td>
			<td>PZN 8473637</td>
			<td></td>
		</tr>
		<tr>
			<td>Preamplifier</td>
			<td>PCB Pieztronics</td>
			<td>480C02</td>
			<td></td>
		</tr>
		<tr>
			<td>RZ6 Multi I/O Processor system (BioSigRZ)</td>
			<td>Tucker-Davis Technologies (TDT)</td>
			<td>RZ6-A-PI</td>
			<td></td>
		</tr>
		<tr>
			<td>0.9% saline (NaCl, sterile)</td>
			<td>B.Braun</td>
			<td>PZN:8609255</td>
			<td></td>
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		<tr>
			<td>SigGenRZ software</td>
			<td>Tucker-Davis Technologies (TDT)</td>
			<td>https://www.tdt.com/</td>
			<td></td>
		</tr>
		<tr>
			<td>Software R (version 3.2.1) + Reshape 2 (Version 1.4.1) + ggplot 2 (version 1.0.1) + datatable (version 1.9.4), + gdata (version 2.13.3), + pastecs (version 1.3.18), + waveslim (version 1.7.5), + MassSpecWavelet (version 1.30.0)</td>
			<td>The R Foundation, R Core Team 2015</td>
			<td>Open Source Software (freely distributable)</td>
			<td></td>
		</tr>
		<tr>
			<td>Sound attenuating cubicle</td>
			<td>Med Associates Inc.</td>
			<td>ENV-018V</td>
			<td></td>
		</tr>
		<tr>
			<td>Standard Pattern Forceps, 12cm and 14.5 cm length</td>
			<td>FST</td>
			<td>11000-12, 11000-14</td>
			<td></td>
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			<td>Leukosilk tape</td>
			<td>BSN medical GmbH &#38; Co. KG</td>
			<td>PZN 00397109</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue Forceps- 1x2 Teeth 12 cm</td>
			<td>FST</td>
			<td>11021-12</td>
			<td></td>
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			<td>Uniprotect ventilated cabinet</td>
			<td>Bioscape</td>
			<td>THF3378</td>
			<td></td>
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			<td>Ventilated cabinet</td>
			<td>Tecniplast</td>
			<td>9AV125P</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylazine (Rompun), 2%</td>
			<td>Bayer Vital GmbH</td>
			<td>PZN 1320422</td>
			<td></td>
		</tr>
	</tbody>
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data acquisition and analysis in brainstem evoked response audiometry in mice

Brainstem evoked response audiometry (BERA) is of central relevance in the clinical neurophysiology. As other evoked potential (EP) techniques, such as visually evoked potentials (VEPs) or somatosensory evoked potentials (SEPs), the auditory evoked potentials (AEPs) are triggered by the repetitive presentation of identical stimuli, the electroencephalographic (EEG) response of which is subsequently averaged resulting in distinct positive (p) and negative (n) deflections. In humans, both the amplitude and the latency of individual peaks can be used to characterize alterations in synchronization and conduction velocity in the underlying neuronal circuitries. Importantly, AEPs are also applied in basic and preclinical science to identify and characterize the auditory function in pharmacological and genetic animal models. Even more, animal models in combination with pharmacological testing are utilized to investigate for potential benefits in the treatment of sensorineural hearing loss (e.g., age- or noise-induced hearing deficits). Here we provide a detailed and integrative description of how to record auditory brainstem-evoked responses (ABRs) in mice using click and tone-burst application. A specific focus of this protocol is on pre-experimental animal housing, anesthesia, ABR recording, ABR filtering processes, automated wavelet-based amplitude growth function analysis, and latency detection.

Click- and tone burst-evoked ABR recordings can be used to evaluate hearing threshold differences, amplitude growth function, and latency comparison. Click-evoked ABRs in the SPL increasing mode are depicted in Figure 1 for controls and two exemplary mutant mouse lines which are deficient for the Cav3.2 T-type voltage-gated Ca2+ channel (i.e., Cav3.2+/- and Cav3.2 null mutants [Cav3.2-/-</...

Watch this Scientific Journal Video about Data Acquisition and Analysis In Brainstem Evoked Response Audiometry In Mice at JoVE.com

Data Acquisition and Analysis In Brainstem Evoked Response Audiometry In Mice

Brainstem evoked response audiometry is an important tool in clinical neurophysiology. Nowadays, brainstem evoked response audiometry is also applied in the basic science and preclinical studies involving both pharmacological and genetic animal models. Here we provide a detailed description of how auditory brainstem responses can be successfully recorded and analyzed in mice.

Brainstem evoked response audiometry (BERA) is of central relevance in the clinical neurophysiology. As other evoked potential (EP) techniques, such as ...

This protocol provides detailed information about how to plan, carry out and analyze click and tone-burst evoked auditory brainstem responses in mice. The main advantage of this technique is that it allows for complex and fast auditory profiling of pharmacological and mutant mouse models. The new insights into alt-er-ed early APP and associated change in the auditory processing in mice and rats can be translated to humans.Therefore, this method is of central importance in the characterization and the phenotyping of auditory, neurological and diseases. This method is most important for the identification of dysacusis, hypoacusis and anacusis. For example, in age related, noise-induced metabolic, congenital and aspi-met-acury hearing loss as well as in hearing deficits due to deformities or malformations, injuries and neoplasms.Users new to the technique, should pay special attention to proper electro-placement and pre-experimental calibration of the system. Visual demonstration of the method is critical to illustrate anesthesia, ABR recording, ABR filtering processes and automated ABR neur-o-lass-es. Begin by turning on the pre-amplifier connected to the microphone, at least five minutes prior to the calibration, to allow for the equilibration of the system.Turn on the oscilloscope. Then position the microphone, connected to a pre-amplifier inside the sound attenuating cubical, to mimic the experimental murine ear. Next, open the commercially available processing and acquisition softwares and program the stimulus protocols for the clicks and tone bursts.Start with the click stimulus entity to analysis and determine click thresholds. Followed by ABR symmetry of the left and right ear. And ABR amplitudes and latencies later on.Next, use the same software to verify tone burst stimulation protocol using Sig-Gen RZ stimulus design software. And checks stimulus settings, under Bio-Sig RZ acquisition software. Program the appropriate frequency range to be tested, depending on the scientific question, and ensure that the frequency ranges to be applied meet the technical capabilities of the loud speaker.For averaging, set the number of sequential acoustics stimuli, either clicks or tone bursts for instance, at 300 times, with a rate of 20 per second;an averaging duration of 25 milliseconds and the amplification factor of the pre-amplifier, 20 times. Next, verify appropriate sampling rate for ABR data acquisition and then pass filter using a six-poll butter-worth filter. Activate the notch filter if necessary.Start the tone burst calibration by selecting the calibration:CAL200K file;within the software, to activate the calibration configuration mode. And choose perimeters according to the experimental conditions. Use the processor system to execute the calibration procedure.Make sure that the technical specifications of the microphone and loud speaker, in terms of sound pressure level or SPL limits, frequency range and distribution harmonize. Then, select and start the pre-defined click stimulation protocol. Run a single click SPL to verify that the spectrum of sound stimuli is analyzed by online fast four-ay transformation of the oscilloscope, matches the requirements.Select and start the pre-defined tone burst stimulation protocol, within the range of one to 42 kilohertz. Confirm the frequency spectrum of the recorded acoustic test stimuli, by using an oscilloscope and online FFT. Finally, complete the tone burst calibration by loading the created calibration file to the tone burst stimulus protocol.Begin by placing the anesthetized mouse inside a sound-attenuating cubicle, lined with acoustical foam. For the recording of monaural brain stem evoked auditory potentials, insert sub-dermal stainless steel electrodes at the vertex, axial of the pin-eye and ventral-lateral of the right or left pinna depending on the ear to be measured. On the other end, for binaural recordings, place the negative electrodes at both the right and left pinna.Position the ground electrode at the hip of the animal. Prior to the insertion, form a hook shape at the tip of the stainless steel electrode that's sub-dermal fixation of the electrodes is guaranteed. And once inserted, properly place the rostrum of the mouse 10 centimeters opposite to the loud speaker.Connect all electrodes to the head stage and check for their impedance. Then, perform impedance measurements of all electrodes, prior to each recording, to verify proper electrode positioning and conductivity. Record ABRs under free field conditions using a single loud speaker.Finally, conduct ABR analysis via automated threshold detection and wave latency analysis to determine positive peaks and negative waves. As a first step in analyzing general hearing performance, click-evoked ABRs for different SPLs, between zero and 90, were investigated using the automated ABR threshold detection system. Potential alterations in ABR threshold levels evoked by different tone burst frequencies were analyzed.In the exemplary mouse lines, Cav3.2 plus, minus and Cav3.2 minus, minus exhibited increased click and tone burst related hearing thresholds compared to controls. Lastly, click-evoked ABR amplitude growth function and ABR wave form latency analysis were carried out via wavelet based approach. The latter allows insight into the possible spacio-temporal influence of the gene of interest on auditory information processing within the inner ear and brainstem.Proper ABR electrode placement in pinna's measurement and system calibration are essential for performing this technique. The auditory approach presented here, can also be used in combination with a telemetry system to analyze complex, mid-latency and late-auditory evoked potentials. This helps in the characterization and in the phenotyping of auditory, neurological and neuro diseases.

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Research

JoVE Journal

Neuroscience

High Density Event-related Potential  Data Acquisition in Cognitive Neuroscience

Functional magnetic resonance imaging (fMRI) is currently the standard method of evaluating brain function in the field of Cognitive Neuroscience, in part because fMRI data acquisition and analysis techniques are readily available. Because fMRI has excellent spatial resolution but poor temporal resolution, this method can only be used to identify the spatial location of brain activity associated with a given cognitive process (and reveals virtually nothing about the time course of brain activity). By contrast, event-related potential (ERP) recording, a method that is used much less frequently than fMRI, has excellent temporal resolution and thus can track rapid temporal modulations in neural activity. Unfortunately, ERPs are under utilized in Cognitive Neuroscience because data acquisition techniques are not readily available and low density ERP recording has poor spatial resolution. In an effort to foster the increased use of ERPs in Cognitive Neuroscience, the present article details key techniques involved in high density ERP data acquisition. Critically, high density ERPs offer the promise of excellent temporal resolution and good spatial resolution (or excellent spatial resolution if coupled with fMRI), which is necessary to capture the spatial-temporal dynamics of human brain function.

Event-related potential (ERP) recording is under utilized in Cognitive Neuroscience because data acquisition techniques are not readily available and this method often has poor spatial resolution. To foster the increased use of ERPs in Cognitive Neuroscience, the present article details key techniques involved in high density ERP data acquisition.

Functional magnetic resonance imaging (fMRI) is currently the standard method of evaluating brain function in the field of Cognitive Neuroscience, in part ...

Basics of Multivariate Analysis in Neuroimaging Data

Multivariate analysis techniques for neuroimaging data have recently received increasing attention as they have many attractive features that cannot be easily realized by the more commonly used univariate, voxel-wise, techniques1,5,6,7,8,9. Multivariate approaches evaluate correlation/covariance of activation across brain regions, rather than proceeding on a voxel-by-voxel basis. Thus, their results can be more easily interpreted as a signature of neural networks. Univariate approaches, on the other hand, cannot directly address interregional correlation in the brain. Multivariate approaches can also result in greater statistical power when compared with univariate techniques, which are forced to employ very stringent corrections for voxel-wise multiple comparisons. Further, multivariate techniques also lend themselves much better to prospective application of results from the analysis of one dataset to entirely new datasets. Multivariate techniques are thus well placed to provide information about mean differences and correlations with behavior, similarly to univariate approaches, with potentially greater statistical power and better reproducibility checks. In contrast to these advantages is the high barrier of entry to the use of multivariate approaches, preventing more widespread application in the community. To the neuroscientist becoming familiar with multivariate analysis techniques, an initial survey of the field might present a bewildering variety of approaches that, although algorithmically similar, are presented with different emphases, typically by people with mathematics backgrounds. We believe that multivariate analysis techniques have sufficient potential to warrant better dissemination. Researchers should be able to employ them in an informed and accessible manner. The current article is an attempt at a didactic introduction of multivariate techniques for the novice. A conceptual introduction is followed with a very simple application to a diagnostic data set from the Alzheimer s Disease Neuroimaging Initiative (ADNI), clearly demonstrating the superior performance of the multivariate approach.

The current article describes the basics of multivariate analysis and contrasts it to the more commonly used voxel-wise univariate analysis. Both types of analysis are applied to a clinical-neuroscience data set. Supplementary split-half simulations show better replication of the multivariate results in independent data sets.

Multivariate analysis techniques for neuroimaging data have recently received increasing attention as they have many attractive features that cannot be ...

Dual Electrophysiological Recordings of Synaptically-evoked Astroglial and Neuronal Responses in Acute Hippocampal Slices

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs through activation of their ion channels and neurotransmitter receptors, and process information in part through activity-dependent release of gliotransmitters. Furthermore, astrocytes constitute the main uptake system for glutamate, contribute to potassium spatial buffering, as well as to GABA clearance. These cells therefore constantly monitor synaptic activity, and are thereby sensitive indicators for alterations in synaptically-released glutamate, GABA and extracellular potassium levels. Additionally, alterations in astroglial uptake activity or buffering capacity can have severe effects on neuronal functions, and might be overlooked when characterizing physiopathological situations or knockout mice. Dual recording of neuronal and astroglial activities is therefore an important method to study alterations in synaptic strength associated to concomitant changes in astroglial uptake and buffering capacities. Here we describe how to prepare hippocampal slices, how to identify stratum radiatum astrocytes, and how to record simultaneously neuronal and astroglial electrophysiological responses. Furthermore, we describe how to isolate pharmacologically the synaptically-evoked astroglial currents.

The preparation of acute brain slices from isolated hippocampi, as well as the simultaneous electrophysiological recordings of astrocytes and neurons in stratum radiatum during stimulation of schaffer collaterals is described. The pharmacological isolation of astroglial potassium and glutamate transporter currents is demonstrated.

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs ...

A Low Cost Setup for Behavioral Audiometry in Rodents

In auditory animal research it is crucial to have precise information about basic hearing parameters of the animal subjects that are involved in the experiments. Such parameters may be physiological response characteristics of the auditory pathway, e.g. via brainstem audiometry (BERA). But these methods allow only indirect and uncertain extrapolations about the auditory percept that corresponds to these physiological parameters. To assess the perceptual level of hearing, behavioral methods have to be used. A potential problem with the use of behavioral methods for the description of perception in animal models is the fact that most of these methods involve some kind of learning paradigm before the subjects can be behaviorally tested, e.g. animals may have to learn to press a lever in response to a sound. As these learning paradigms change perception itself 1,2 they consequently will influence any result about perception obtained with these methods and therefore have to be interpreted with caution. Exceptions are paradigms that make use of reflex responses, because here no learning paradigms have to be carried out prior to perceptual testing. One such reflex response is the acoustic startle response (ASR) that can highly reproducibly be elicited with unexpected loud sounds in na&iuml;ve animals. This ASR in turn can be influenced by preceding sounds depending on the perceptibility of this preceding stimulus: Sounds well above hearing threshold will completely inhibit the amplitude of the ASR; sounds close to threshold will only slightly inhibit the ASR. This phenomenon is called pre-pulse inhibition (PPI) 3,4, and the amount of PPI on the ASR gradually depends on the perceptibility of the pre-pulse. PPI of the ASR is therefore well suited to determine behavioral audiograms in na&iuml;ve, non-trained animals, to determine hearing impairments or even to detect possible subjective tinnitus percepts in these animals. In this paper we demonstrate the use of this method in a rodent model (cf. also ref. 5), the Mongolian gerbil (Meriones unguiculatus), which is a well know model species for startle response research within the normal human hearing range (e.g. 6).

A fast and inexpensive method for the behavioral determination of hearing parameters like hearing thresholds, hearing impairments or phantom perceptions (subjective tinnitus) is described. It uses pre-pulse inhibition of the acoustic startle response and can be easily implemented in a personal computer using a programmable AD/DA-converter and a piezo sensor.

In auditory animal research it is crucial to have precise information about basic hearing parameters of the animal subjects that are involved in the ...

TIRFM and pH-sensitive GFP-probes to Evaluate Neurotransmitter Vesicle Dynamics in SH-SY5Y Neuroblastoma Cells: Cell Imaging and Data Analysis

Synaptic vesicles release neurotransmitters at chemical synapses through a dynamic cycle of fusion and retrieval. Monitoring synaptic activity in real time and dissecting the different steps of exo-endocytosis at the single-vesicle level are crucial for understanding synaptic functions in health and disease.
Genetically-encoded pH-sensitive probes directly targeted to synaptic vesicles and Total Internal Reflection Fluorescence Microscopy (TIRFM) provide the spatio-temporal resolution necessary to follow vesicle dynamics. The evanescent field generated by total internal reflection can only excite fluorophores placed in a thin layer (&#60;150 nm) above the glass cover on which cells adhere, exactly where the processes of exo-endocytosis take place. The resulting high-contrast images are ideally suited for vesicles tracking and quantitative analysis of fusion events.
In this protocol, SH-SY5Y human neuroblastoma cells are proposed as a valuable model for studying neurotransmitter release at the single-vesicle level by TIRFM, because of their flat surface and the presence of dispersed vesicles. The methods for growing SH-SY5Y as adherent cells and for transfecting them with synapto-pHluorin are provided, as well as the technique&#160;to perform TIRFM and imaging. Finally, a strategy aiming to select, count, and analyze fusion events at whole-cell and single-vesicle levels is presented.
To validate the imaging procedure and data analysis approach, the dynamics of pHluorin-tagged vesicles are&#160;analyzed under resting and stimulated (depolarizing potassium concentrations) conditions. Membrane depolarization increases the frequency of fusion events and causes a parallel raise of the net fluorescence signal recorded in whole cell. Single-vesicle analysis reveals modifications of fusion-event behavior (increased peak height and width). These data suggest that potassium depolarization not only induces a massive neurotransmitter release but also modifies the mechanism of vesicle fusion and recycling.
With the appropriate fluorescent probe, this technique can be employed in different cellular systems to dissect the mechanisms of constitutive and stimulated secretion.

This paper provides a method for investigating neurotransmitter vesicle dynamics in neuroblastoma cells, using a synaptobrevin2-pHluorin construct and Total Internal Reflection Fluorescence Microscopy. The strategy developed for image processing and data analysis is also reported.

Synaptic vesicles release neurotransmitters at chemical synapses through a dynamic cycle of fusion and retrieval. Monitoring synaptic activity in real ...

A Simple and Inexpensive Method for Determining Cold Sensitivity and Adaptation in Mice

Cold hypersensitivity is a serious clinical problem, affecting a broad subset of patients and causing significant decreases in quality of life. The cold plantar assay allows the objective and inexpensive assessment of cold sensitivity in mice, and can quantify both analgesia and hypersensitivity. Mice are acclimated on a glass plate, and a compressed dry ice pellet is held against the glass surface underneath the hindpaw. The latency to withdrawal from the cooling glass is used as a measure of cold sensitivity.
Cold sensation is also important for survival in regions with seasonal temperature shifts, and in order to maintain sensitivity animals must be able to adjust their thermal response thresholds to match the ambient temperature. The Cold Plantar Assay (CPA) also allows the study of adaptation to changes in ambient temperature by testing the cold sensitivity of mice at temperatures ranging from 30 &#176;C to 5 &#176;C. Mice are acclimated as described above, but the glass plate is cooled to the desired starting temperature using aluminum boxes (or aluminum foil packets) filled with hot water, wet ice, or dry ice. The temperature of the plate is measured at the center using a filament T-type thermocouple probe. Once the plate has reached the desired starting temperature, the animals are tested as described above.
This assay allows testing of mice at temperatures ranging from innocuous to noxious. The CPA yields unambiguous and consistent behavioral responses in uninjured mice and can be used to quantify both hypersensitivity and analgesia. This protocol describes how to use the CPA to measure cold hypersensitivity, analgesia, and adaptation in mice.

The Cold Plantar Assay (CPA) measures cold responsiveness between 30 &#176;C and 5 &#176;C, and can also measure cold adaptation. This protocol describes how to use the CPA to measure cold hypersensitivity, analgesia, and adaptation in mice.

Cold hypersensitivity is a serious clinical problem, affecting a broad subset of patients and causing significant decreases in quality of life.  The cold ...

Electroretinogram Analysis of the Visual Response in Zebrafish Larvae

The electroretinogram (ERG) is a noninvasive electrophysiological method for determining retinal function. Through the placement of an electrode on the surface of the cornea, electrical activity generated in response to light can be measured and used to assess the activity of retinal cells in vivo. This manuscript describes the use of the ERG to measure visual function in zebrafish. Zebrafish have long been utilized as a model for vertebrate development due to the ease of gene suppression by morpholino oligonucleotides and pharmacological manipulation. At 5-10 dpf, only cones are functional in the larval retina. Therefore, the zebrafish, unlike other animals, is a powerful model system for the study of cone visual function in vivo. This protocol uses standard anesthesia, micromanipulation and stereomicroscopy protocols that are common in laboratories that perform zebrafish research. The outlined methods make use of standard electrophysiology equipment and a low light camera to guide the placement of the recording microelectrode onto the larval cornea. Finally, we demonstrate how a commercially available ERG stimulator/recorder originally designed for use with mice can easily be adapted for use with zebrafish. ERG of larval zebrafish provides an excellent method of assaying cone visual function in animals that have been modified by morpholino oligonucleotide injection as well as newer genome engineering techniques such as Zinc Finger Nucleases (ZFNs), Transcription Activator-Like Effector Nucleases (TALENs), and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9, all of which have greatly increased the efficiency and efficacy of gene targeting in zebrafish. In addition, we take advantage of the ability of pharmacological agents to penetrate zebrafish larvae to evaluate the molecular components that contribute to the photoresponse. This protocol outlines a setup that can be modified and used by researchers with various experimental goals.

We present a method for the electroretinographic (ERG) analysis of zebrafish larvae utilizing micromanipulation and electroretinography techniques. This is a simple and straightforward method for assaying visual function of zebrafish larvae in vivo.

The electroretinogram (ERG) is a noninvasive electrophysiological method for determining retinal function. Through the placement of an electrode on the ...

Auditory Brainstem Response and Outer Hair Cell Whole-cell Patch Clamp Recording in Postnatal Rats

The outer hair cell is one of the two types of sensory hair cells in the mammalian cochlea. They alter their cell length with the receptor potential to amplify the weak vibration of low-level sound signal. The morphology and electrophysiological property of outer hair cells (OHCs) develop in early postnatal ages. The maturation of outer hair cell may contribute to the development of the auditory system. However, the process of OHCs development is not well studied. This is partly because of the difficulty to measure their function by an electrophysiological approach. With the purpose of developing a simple method to address the above issue, here we describe a step-by-step protocol to study the function of OHCs in acutely dissociated cochlea from postnatal rats. With this method, we can evaluate the cochlear response to pure tone stimuli and examine the expression level and function of the motor protein prestin in OHCs. This method can also be used to investigate the inner hair cells (IHCs).

This protocol describes methods to record the auditory brainstem response from postnatal rat pups. To examine the functional development of outer hair cells, the experimental procedure of whole-cell patch clamp recording in isolated outer hair cells is described step-by-step.

The outer hair cell is one of the two types of sensory hair cells in the mammalian cochlea. They alter their cell length with the receptor potential to ...

Cochlear Implant Surgery and Electrically-evoked Auditory Brainstem Response Recordings in C57BL/6 Mice

Cochlear implants (CIs) are neuroprosthetic devices that can provide a sense of hearing to deaf people. However, a CI cannot restore all aspects of hearing. Improvement of the implant technology is needed if CI users are to perceive music and perform in more natural environments, such as hearing out a voice with competing talkers, reflections, and other sounds. Such improvement requires experimental animals to better understand the mechanisms of electric stimulation in the cochlea and its responses in the whole auditory system. The mouse is an increasingly attractive model due to the many genetic models available. However, the limited use of this species as a CI model is mainly due to the difficulty of implanting small electrode arrays. More details about the surgical procedure are therefore of great interest to expand the use of mice in CI research.
In this report, we describe in detail the protocol for acute deafening and cochlear implantation of an electrode array in the C57BL/6 mouse strain. We demonstrate the functional efficacy of this procedure with electrically-evoked auditory brainstem response (eABR) and show examples of facial nerve stimulation. Finally, we also discuss the importance of including a deafening procedure when using a normally hearing animal. This mouse model provides a powerful opportunity to study genetic and neurobiological mechanisms that would be of relevance for CI users.

Animal models of cochlear implants can advance knowledge of the technological bases of treating permanent sensorineural hearing loss with electrical stimulation. This study presents a surgical protocol for acute deafening and cochlear implantation of an electrode array in mice as well as the functional assessment with auditory brainstem response.

Cochlear implants (CIs) are neuroprosthetic devices that can provide a sense of hearing to deaf people. However, a CI cannot restore all aspects of ...

Reliable Acquisition of Electroencephalography Data during Simultaneous Electroencephalography and Functional MRI

Simultaneous electroencephalography (EEG) and functional magnetic resonance imaging (fMRI), EEG-fMRI, combines the complementary properties of scalp EEG (good temporal resolution) and fMRI (good spatial resolution) to measure neuronal activity during an electrographic event, through hemodynamic responses known as blood-oxygen-level-dependent (BOLD) changes. It is a non-invasive research tool that is utilized in neuroscience research and is highly beneficial to the clinical community, especially for the management of neurological diseases, provided that proper equipment and protocols are administered during data acquisition. Although recording EEG-fMRI is apparently straightforward, the correct preparation, especially in placing and securing the electrodes, is not only important for safety but is also critical in ensuring the reliability and analyzability of the EEG data obtained. This is also the most experience-demanding part of the preparation. To address these issues, a straightforward protocol that ensures data quality was developed. This article provides a step-by-step guide for acquiring reliable EEG data during EEG-fMRI using this protocol that utilizes readily available medical products. The presented protocol can be adapted to different applications of EEG-fMRI in research and clinical settings, and may be beneficial to both inexperienced and expert operators.

This article provides a straightforward protocol for acquiring good quality electroencephalography (EEG) data during simultaneous EEG and functional magnetic resonance imaging by utilizing readily available medical products.

Simultaneous electroencephalography (EEG) and functional magnetic resonance imaging (fMRI), EEG-fMRI, combines the complementary properties of scalp EEG ...