This work was supported by the Interdisciplinary Center for Clinical Research (IZKF, project E7) at the University Hospital of the University of Erlangen-Nuremberg.

1. Setup Assembling and Software Programming
<ol>
	<li>Install a D/A card in a Personal Computer (e.g.: NI PCI 6229, National Instruments) and connect it to a breakout-box (e.g.: BNC-2110, National Instruments), both should support at least one input and one output channel with a sampling rate of at least 44.1 kHz each.</li>
	<li>Connect the output of the breakout-box via BNC cable to a sound amplifier (e.g: AMP75 wideband power amplifier, Thomas Wulf).</li>
	<li>Install an infrared webcam (e.g.: Grand IP Camera Pro, GrandTec Electronics) for animal surveillance in darkness.</li>
	<li>Install an Integrated Development Environment (e.g.: Matlab) to implement a program as defined by the flowchart given in Figure 1. A version running on Matlab 2007b may be obtained free of charge from the corresponding author.</li>
	<li>Within a soundproof chamber install a loudspeaker on a table. Connect the speaker to the sound amplifier.</li>
	<li>Install a piezo sensor (e.g. force sensor FSG15N1A, Honeywell) on top of an insulating board, support it with power and connect it via BNC cable to the input of the breakout-box; ground the sensor signal.</li>
	<li>Build a measuring chamber from an acrylic glass tube adjusted to the size of the rodent to measure (for gerbils e.g.: length 15 cm, inner diameter 4.3 cm, outer diameter 4.8 cm). Fix a grate with a mesh width of 0.5 mm to the front of the tube and a cushioned door with a lock mechanism (e.g., a hook) to the rear. The fixation can be done with hot glue, for the door only the hinge and the bail for the hook is fixed to the tube.</li>
	<li>Attach foam plastic feet that fit the measuring tube dimensions on the insulating sensor board. Feet should support the tube under the front and rear end and lift the tube to the sensor level. Make sure that there is light contact between the piezo sensor and the measuring chamber.</li>
	<li>Fix the sensor board with the measuring chamber centered in front of the speaker and put a microphone for sound control next to it (e.g. B&amp;K Type 2669 / B&amp;K Type 4190 connected to measuring amplifier B&amp;K Type 2610, all: Bruel and Kjaer) at the level of the animals head so that it is not interfering with the tube.</li>
	<li>Note that the quality of your audiometric data will depend on the quality of your sound system. In any case, use the microphone and measuring amplifier to determine the frequency transfer function of your system prior to your first experiments and include a routine in your software to correct for this frequency transfer function to make the spectral output of your speaker flat.</li>
	<li>Align the webcam with the setup so that one can monitor animal behavior.</li>
</ol>
2. Behavioral Determination of Hearing Thresholds (Audiograms)
<ol>
	<li>Take the animal from its home cage and put it head first in the tube; close the door.</li>
	<li>Put the tube on the foam feet and the sensor. Switch off any light and close the door to the chamber. The chamber itself is not air conditioned but has the temperature, humidity and other environmental variables of the surrounding laboratory. Air change by ventilators is not advisable due to the induced noise but the volume of the chamber is supporting oxygen for the animal for many hours. Wait 15 min to allow the animal to get accustomed to the setup. The acclimatization time is beneficial for the animal as it can calm down at its own pace and get used to the tube. On the other hand, one does not see any differences in behavior during the acclimatization time over several session, which indicates that an extra session for familiarizing the animal with the tube and the chamber prior to the experiment is not needed.</li>
	<li>Start the program and define the parameters for stimulation (cf. also ref. 5): Stimuli consist of pure tones with different frequencies. The startle stimulus must be presented with a sound pressure level high enough to reproducibly elicit a startle response. In our lab we use intensities of 105 dB SPL (stimulus duration 6 ms including 2 ms cosine-squared rise and fall ramps) to elicit startle responses in Mongolian gerbils. The test stimuli preceding the startle stimuli are presented at varying frequencies and intensities in the range to be tested, usually from below hearing threshold to levels well above threshold and with frequencies covering the whole audible range of the species. Frequency and duration of the startle and the test stimulus are matched in each trial, the interstimulus interval between startle and test stimulus is set to 100 ms. Use at least 15 repetitions with randomized interstimulus intervals of 10 &plusmn; 2.5 sec for each frequency and intensity combination of the test stimuli (cf. Figure 2A, left). Subsequent test stimuli can be presented in either randomized or non-randomized order. If you are using a non-randomized approach (e.g., one test stimulus level fixed for all tested frequencies) allow 5 min of recovery between different stimulus sets. Note that the determined absolute threshold will depend on the randomization of the stimuli, but possible relative shifts of threshold will not (e.g. after acoustic trauma, cf. ref. 5).</li>
	<li>Before analyzing the data, remove invalid trials from dataset (e.g., trials where the animal moved before the startle stimulus; cf. Figure 2B).</li>
	<li>Within a time window of the first 50 ms after the startle stimulus, calculate the response amplitude (peak to peak between the first maximum to the first minimum of the response) and response latency (time from stimulus start to response onset) of each single trial.</li>
	<li>Fit a Boltzmann-function to the complete response amplitude data set of one frequency sorted for prestimulus intensity for all valid single trials. The 50% point of the Boltzmann-function 7 indicates the hearing threshold for this stimulation frequency.</li>
</ol>
3. Acoustic Trauma and Quantification of Hearing Impairment
<ol>
	<li>Prepare ketamine-xylazine-anesthesia with a mixture of ketamine hydrochloride: 96 mg/kg (Ketamin-ratiopharm, Ratiopharm); xylazine hydrochloride: 4 mg/kg (Rompun 2%, Bayer); atropine sulfate: 1 mg/kg (Atropinsulfat, B. Braun Melsungen AG) and physiological NaCl-solution (Berlin-Chemie AG, Berlin) with a ratio of 9:1:2:8.</li>
	<li>Inject the animal with 3 ml/kg of anesthesia subcutaneous. Wait until the animal is deeply anesthetized (ca. 5 min, check reflexes, e.g., use the pedal withdrawal reflex). To maintain anesthesia during measurements, continuously inject the anesthetic solution at a rate of 3 ml/kg/hr using a syringe pump. Control vital signs with appropriate equipment (e.g., breathing via camera) and keep the animal warm by placing it on a warming pad.</li>
	<li>Induce an acoustic trauma, e.g. using a loud pure tone: e.g. 2 kHz at 115dB SPL for 75 min.</li>
	<li>After the end of trauma stop the syringe pump and let the animal awake in a wake-up cage on a warming pad at a quiet place. Check regularly during the awakening phase if the vital signs are stable. Put the animal in its home cage only when it is completely awake. Let the animal recover from the anesthesia (at least 2 days) and rest in its home cage.</li>
	<li>Perform 2.1 to 2.6 again. Compare the hearing thresholds before and after the acoustic trauma by calculating the percentage of hearing loss for each frequency. After the end of all experiments painlessly euthanize the animal.</li>
</ol>
4. Test for Acoustic Phantom Perception (Subjective tinnitus)
<ol>
	<li>Perform these measurements before and after the acoustic trauma.</li>
	<li>Follow 2.1 to 2.2 if the animal is not already in the setup.</li>
	<li>A number of stimulation paradigms can be used to test rodents for subjective tinnitus. The ratio in all these approaches is to test for the salience of a silent gap within a background noise. If the gap is perceived by the animals, it can be used as a test stimulus to reduce a startle response analogously to the procedure described in 2. If the animal suffers from tinnitus (that is likely to develop after acoustic trauma), this tinnitus will be perceived within the silent gap and therefore makes the gap less salient. The effect of the gap on the startle response consequently will be weaker in tinnitus animals compared to healthy controls (reduced PPI, cf. 8). This percept is tested with two slightly different protocols.</li>
	<li>In the first subjective tinnitus paradigm presented here (Figure 2A, center, cf. 9) use the following parameters for stimulation: startle sound intensity 105 dB SPL with frequencies from 1 to 16 kHz in 1 octave steps, stimulus length 6 ms including 2 ms cosine-squared rise and fall ramps. Present a white noise background of 50 dB SPL during the experiment, either with or without a 15 ms gap that precedes the startle stimulus by 100 ms; present at least 15 trials for each frequency and gap-condition. If using a non-randomized approach, allow 5 min recovery between the different stimulus sets. Use of different startle stimulus frequencies will give a rough estimate of the perceived tinnitus frequency.</li>
	<li>As a second subjective tinnitus paradigm (Figure 2A, right) you may use the following stimulation parameters: startle sound intensity 105 dB SPL, double click stimulus with 0.1 ms duration per click and 0.1 ms between the clicks, the second click having the inverted direction compared to the first. Present a bandpass filtered noise background of 50 dB SPL with a gaussian filter width of 0.5 octaves and center frequencies ranging from 1 to 16 kHz in octave steps. Present this noise either with or without a 15 ms gap that precedes the startle stimulus by 100 ms; present at least 15 trials for each frequency and gap-condition. If using a non-randomized approach, allow 5 min recovery between the different stimulus sets. Use of different center frequencies of the bandpass background noise will give a rough estimate of the perceived tinnitus frequency.</li>
	<li>Follow 2.4 to 2.5; normalize all acquired data with a reference data set for each tested frequency and each trauma condition, i.e., before and after an acoustic trauma. This reference is the response amplitude to the pure tone startle stimulus without any prestimulus (cf. 2.3). The frequency is determined either by the pure tone or the center frequency of the bandpass filtered noise. Calculate the mean response of each reference and normalize each calculated response amplitude by dividing it through its reference.</li>
	<li>Calculate PPI by dividing the normalized response amplitudes of the gap condition through the mean of the normalized no-gap condition of each tested frequency.</li>
	<li>Calculate the PPI change after the trauma in percent for each tested frequency.</li>
</ol>
The startle responses of animals are easy to generate and to analyze. Figure 2B gives an overview of a typical result of one animal stimulated with a pure tone of 105 dB SPL without any prestimulus for 15 times. The majority of the trials are valid and invalid trials are easy to recognize (trials marked by red square). The response amplitudes and latencies are calculated only from valid trials.
A typical behavioral threshold change is given in Figure 3A. The audiogram of an exemplary animal acquired with the method described in 2 is given before (blue) and after (red) an acoustic trauma at 2 kHz (yellow area). A clear hearing loss is shown specifically at 2 kHz. The responses related to a subjective tinnitus percept can be seen in Figure 3B, the normalized response amplitudes of the same animal as above are shown exemplarily for stimulations one octave below and above the trauma as described in 4.5. The comparison of the responses to stimuli with and without gap before (blue) and after the trauma (red) allows an interpretation of a possible tinnitus percept. Below the trauma frequency no change of response pattern can be found while above the trauma the effect of the gap vanished after the trauma, indicating a mispercept at this frequency.
<img alt="Figure 1" src="/files/ftp_upload/4433/4433fig1.jpg" /> 
Figure 1. Flow diagram of the program used to acquire the behavioral thresholds and subjective tinnitus data. Note that this is only a simplified version of the program code. Abbreviations: GUI - graphical user interface; ISI - inter stimulus interval.
<img alt="Figure 2" src="/files/ftp_upload/4433/4433fig2.jpg" /> 
Figure 2. Auditory startle response (ASR) stimuli. A Schemes of the three different stimulation protocols used. Left panel: pre-pulse inhibition (PPI) of the ASR measured without any pure tone test stimulus before (green) the startle tone (red); the response period is depicted in blue. Center panel: gap / noise paradigm with pure tone startle stimulus presentation of different frequencies on a white noise background. Right panel: gap / noise paradigm with click startle stimulus presentation on bandpass filtered background of different center frequencies. B Exemplary auditory startle responses of 15 trials recorded with the threshold paradigm without any prestimulus at 1 kHz stimulation frequency. Three trials are counted invalid (red squares) as the animal moved already before the stimulation onset.
<img alt="Figure 3" src="/files/ftp_upload/4433/4433fig3.jpg" /> 
Figure 3. Exemplary results of ASR in one animal. A Behavioral threshold before (blue) and after (red) the acoustic trauma at 2 kHz (yellow area). The thresholds are calculated from the responses to the PPI modulated ASR protocol using the Boltzmann-function turning point as threshold value. Note that the hearing loss at 2 kHz amounts to more than 66%, while farer away from the trauma frequency one often can see even improvement of hearing thresholds. B Normalized response amplitudes (open circles: single trials, filled circles: means, whiskers: standard deviation) during stimulation with the gap / noise click ASR protocol (4.5) for 1 and 4 kHz center frequencies. Responses are sorted for trials without and with gap in the noise before and after the trauma at 2 kHz. Only at 4 kHz the effect of the gap vanishes after the trauma which indicates a subjective tinnitus percept around this frequency.

<ol>
	<li>Brown, M., Irvine, D. R., Park, V. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perceptual+learning+on+an+auditory+frequency+discrimination+task+by+cats:+association+with+changes+in+primary+auditory+cortex.">Perceptual learning on an auditory frequency discrimination task by cats: association with changes in primary auditory cortex.</a> Cereb. Cortex. 14, 952-965 (2004).</li><li>Ohl, F. W., Scheich, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Learning-induced+plasticity+in+animal+and+human+auditory+cortex.">Learning-induced plasticity in animal and human auditory cortex.</a> Curr. Opin. Neurobiol. 15, 470-477 (2005).</li><li>Koch, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+neurobiology+of+startle.">The neurobiology of startle.</a> Prog. Neurobiol. 59, 107-128 (1999).</li><li>Larrauri, J., Schmajuk, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prepulse+inhibition+mechanisms+and+cognitive+processes:+a+review+and+model.">Prepulse inhibition mechanisms and cognitive processes: a review and model.</a> EXS. 98, 245-278 (2006).</li><li>Walter, M., Tziridis, K., Ahlf, S., Schulze, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Context+dependent+auditory+thresholds+determined+by+brainstem+audiometry+and+prepulse+inhibition+in+Mongolian+gerbils.">Context dependent auditory thresholds determined by brainstem audiometry and prepulse inhibition in Mongolian gerbils.</a> Open Journal of Acoustics. 2, 34-49 (2012).</li><li>Gaese, B. H., Nowotny, M., Pilz, P. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acoustic+startle+and+prepulse+inhibition+in+the+Mongolian+gerbil.">Acoustic startle and prepulse inhibition in the Mongolian gerbil.</a> Physiol. Behav. 98, 460-466 (2009).</li><li>Fechter, L. D., Sheppard, L., Young, J. S., Zeger, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensory+threshold+estimation+from+a+continuously+graded+response+produced+by+reflex+modification+audiometry.">Sensory threshold estimation from a continuously graded response produced by reflex modification audiometry.</a> J. Acoust. Soc. Am. 84, 179-185 (1988).</li><li>Turner, J. G., Parrish, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gap+detection+methods+for+assessing+salicylate-induced+tinnitus+and+hyperacusis+in+rats.">Gap detection methods for assessing salicylate-induced tinnitus and hyperacusis in rats.</a> Am. J. Audiol. 17, 185-192 (2008).</li><li>Campeau, S., Davis, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fear+potentiation+of+the+acoustic+startle+reflex+using+noises+of+various+spectral+frequencies+as+conditioned+stimuli.">Fear potentiation of the acoustic startle reflex using noises of various spectral frequencies as conditioned stimuli.</a> Animal Learning &#38; Behavior. 20, 177-186 (1992).</li><li>Young, J. S., Fechter, L. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reflex+inhibition+procedures+for+animal+audiometry:+a+technique+for+assessing+ototoxicity.">Reflex inhibition procedures for animal audiometry: a technique for assessing ototoxicity.</a> J. Acoust. Soc. Am. 73, 1686-1693 (1983).</li><li>Turner, J. G., Brozoski, T. J., Bauer, C. A., Parrish, J. L., Myers, K., Hughes, L. F., Caspary, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gap+detection+deficits+in+rats+with+tinnitus:+a+potential+novel+screening+tool.">Gap detection deficits in rats with tinnitus: a potential novel screening tool.</a> Behav. Neurosci. 120, 188-195 (2006).</li><li>Turner, J., Larsen, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relationship+between+noise+exposure+stimulus+properties+and+tinnitus+in+rats:+Results+of+a+12-month+longitudinal+study.">Relationship between noise exposure stimulus properties and tinnitus in rats: Results of a 12-month longitudinal study.</a> ARO. Abs. 594, (2012).</li><li>Turner, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavioral+measures+of+tinnitus+in+laboratory+animals.">Behavioral measures of tinnitus in laboratory animals.</a> Prog. Brain Res. 166, 147-156 (2007).</li><li>Engineer, N. D., Riley, J. R., Seale, J. D., Vrana, W. A., Shetake, J. A., Shetake, J. A., Sudanagunta, S. P., Borland, M. S., Kilgard, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reversing+pathological+neural+activity+using+targeted+plasticity.">Reversing pathological neural activity using targeted plasticity.</a> Nature. 470, 101-104 (2011).</li></ol>

No conflicts of interest declared.

We present a cheap and easy to build setup for audiometric measurements in rodents based on pre-pulse inhibition of acoustic startle responses that can be used to determine behavioral hearing thresholds (= audiograms 10) and auditory phantom percepts like subjective tinnitus 11. Especially the latter measurements are in the focus of several recent reports 8,12,13,14 and can be seen as one prerequisite for electrophysiological investigations of the neuronal mechanisms underlying this disea...

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).

Watch this Scientific Journal Video about A Low Cost Setup for Behavioral Audiometry in Rodents at JoVE.com

A Low Cost Setup for Behavioral Audiometry in Rodents

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 ...

a-low-cost-setup-for-behavioral-audiometry-in-rodents

Research

JoVE Journal

Neuroscience

12.6K Views.  University of Erlangen-Nuremberg. 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.

Video: A Low Cost Setup for Behavioral Audiometry in Rodents

A General Method for Evaluating Incubation of Sucrose Craving in Rats

For someone on a food-restricted diet, food craving in response to food-paired cues may serve as a key behavioral transition point between abstinence and relapse to food taking 1. Food craving conceptualized in this way is akin to drug craving in response to drug-paired cues. A rich literature has been developed around understanding the behavioral and neurobiological determinants of drug craving; we and others have been focusing recently on translating techniques from basic addiction research to better understand addiction-like behaviors related to food 2-4.
As done in previous studies of drug craving, we examine sucrose craving behavior by utilizing a rat model of relapse. In this model, rats self-administer either drug or food in sessions over several days. In a session, lever responding delivers the reward along with a tone+light stimulus. Craving behavior is then operationally defined as responding in a subsequent session where the reward is not available. Rats will reliably respond for the tone+light stimulus, likely due to its acquired conditioned reinforcing properties 5. This behavior is sometimes referred to as sucrose seeking or cue reactivity. In the present discussion we will use the term "sucrose craving" to subsume both of these constructs.
In the past decade, we have focused on how the length of time following reward self-administration influences reward craving. Interestingly, rats increase responding for the reward-paired cue over the course of several weeks of a period of forced-abstinence. This "incubation of craving" is observed in rats that have self-administered either food or drugs of abuse 4,6. This time-dependent increase in craving we have identified in the animal model may have great potential relevance to human drug and food addiction behaviors.
Here we present a protocol for assessing incubation of sucrose craving in rats. Variants of the procedure will be indicated where craving is assessed as responding for a discrete sucrose-paired cue following extinction of lever pressing within the sucrose self-administration context (Extinction without cues) or as responding for sucrose-paired cues in a general extinction context (Extinction with cues).

Responding for food or drug-paired cues increases over the course of a period of abstinence and this may relate to an increased susceptibility to relapse behaviors. Here we detail a procedure for evaluating this "incubation of craving" in rats that have self-administered sucrose.

For someone on a food-restricted diet, food craving in response to food-paired cues may serve as a key behavioral transition point between abstinence and ...

Large-scale Recording of Neurons by Movable Silicon Probes in Behaving Rodents

A major challenge in neuroscience is linking behavior to the collective activity of neural assemblies. Understanding of input-output relationships of neurons and circuits requires methods with the spatial selectivity and temporal resolution appropriate for mechanistic analysis of neural ensembles in the behaving animal, i.e. recording of representatively large samples of isolated single neurons. Ensemble monitoring of neuronal activity has progressed remarkably in the past decade in both small and large-brained animals, including human subjects1-11. Multiple-site recording with silicon-based devices are particularly effective because of their scalability, small volume and geometric design.
Here, we describe methods for recording multiple single neurons and local field potential in behaving rodents, using commercially available micro-machined silicon probes with custom-made accessory components. There are two basic options for interfacing silicon probes to preamplifiers: printed circuit boards and flexible cables. Probe supplying companies (<a href="http://www.neuronexustech.com/" >http://www.neuronexustech.com/</a>; <a href="http://www.sbmicrosystems.com/">http://www.sbmicrosystems.com/</a>; <a href="http://www.acreo.se/">http://www.acreo.se/</a>) usually provide the bonding service and deliver probes bonded to printed circuit boards or flexible cables. Here, we describe the implantation of a 4-shank, 32-site probe attached to flexible polyimide cable, and mounted on a movable microdrive. Each step of the probe preparation, microdrive construction and surgery is illustrated so that the end user can easily replicate the process.

We describe methods for large-scale recording of multiple single units and local field potential in behaving rodents with silicon probes. Drive fabrication, probe attachment to the drive and probe implantation processes are illustrated in sufficient details for easy replication.

A major challenge in neuroscience is linking behavior to the collective activity of neural assemblies. Understanding of input-output relationships of ...

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.

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

Recording and Analysis of Circadian Rhythms in Running-wheel Activity in Rodents

When rodents have free access to a running wheel in their home cage, voluntary use of this wheel will depend on the time of day1-5. Nocturnal rodents, including rats, hamsters, and mice, are active during the night and relatively inactive during the day. Many other behavioral and physiological measures also exhibit daily rhythms, but in rodents, running-wheel activity serves as a particularly reliable and convenient measure of the output of the master circadian clock, the suprachiasmatic nucleus (SCN) of the hypothalamus. In general, through a process called entrainment, the daily pattern of running-wheel activity will naturally align with the environmental light-dark cycle (LD cycle; e.g. 12 hr-light:12 hr-dark). However circadian rhythms are endogenously generated patterns in behavior that exhibit a ~24 hr period, and persist in constant darkness. Thus, in the absence of an LD cycle, the recording and analysis of running-wheel activity can be used to determine the subjective time-of-day. Because these rhythms are directed by the circadian clock the subjective time-of-day is referred to as the circadian time (CT). In contrast, when an LD cycle is present, the time-of-day that is determined by the environmental LD cycle is called the zeitgeber time (ZT).
Although circadian rhythms in running-wheel activity are typically linked to the SCN clock6-8, circadian oscillators in many other regions of the brain and body9-14 could also be involved in the regulation of daily activity rhythms. For instance, daily rhythms in food-anticipatory activity do not require the SCN15,16 and instead, are correlated with changes in the activity of extra-SCN oscillators17-20. Thus, running-wheel activity recordings can provide important behavioral information not only about the output of the master SCN clock, but also on the activity of extra-SCN oscillators. Below we describe the equipment and methods used to record, analyze and display circadian locomotor activity rhythms in laboratory rodents.

Circadian rhythms in voluntary wheel-running activity in mammals are tightly coupled to the molecular oscillations of a master clock in the brain. As such, these daily rhythms in behavior can be used to study the influence of genetic, pharmacological, and environmental factors on the functioning of this circadian clock.

When rodents have free access to a running wheel in their home cage, voluntary use of this wheel will depend on the time of day1-5. Nocturnal rodents, ...

A Low-cost Method for Analyzing Seizure-like Activity and Movement in Drosophila

Video tracking systems have been used widely to analyze Drosophila melanogaster movement and detect various abnormalities in locomotive behavior. While these systems can provide a wealth of behavioral information, the cost and complexity of these systems can be prohibitive for many labs. We have developed a low-cost assay for measuring locomotive behavior and seizure movement in D. melanogaster. The system uses a web-cam to capture images that can be processed using a combination of inexpensive and free software to track the distance moved, the average velocity of movement and the duration of movement during a specified time-span. To demonstrate the utility of this system, we examined a group of D. melanogaster mutants, the Bang-sensitive (BS) paralytics, which are 3-10 times more susceptible to seizure-like activity (SLA) than wild type flies. Using this novel system, we were able to detect that the BS mutant bang senseless (bss) exhibits lower levels of exploratory locomotion in a novel environment than wild type flies. In addition, the system was used to identify that the drug metformin, which is commonly used to treat type II diabetes, reduces the intensity of SLA in the BS mutants.

A Low-cost Method for Analyzing Seizure-like Activity and Movement in Drosophila

Using a webcam and a combination of free and inexpensive software programs, locomotive patterns in Drosophila melanogaster can be analyzed to detect differences in the speed, distance, and time of various motor activities.

Video tracking systems have been used widely to analyze Drosophila melanogaster movement and detect various abnormalities in locomotive behavior. While ...

A Human-machine-interface Integrating Low-cost Sensors with a Neuromuscular Electrical Stimulation System for Post-stroke Balance Rehabilitation

A stroke is caused when an artery carrying blood from heart to an area in the brain bursts or a clot obstructs the blood flow to brain thereby preventing delivery of oxygen and nutrients. About half of the stroke survivors are left with some degree of disability. Innovative methodologies for restorative neurorehabilitation are urgently required to reduce long-term disability. The ability of the nervous system to reorganize its structure, function and connections as a response to intrinsic or extrinsic stimuli is called neuroplasticity. Neuroplasticity is involved in post-stroke functional disturbances, but also in rehabilitation. Beneficial neuroplastic changes may be facilitated with non-invasive electrotherapy, such as neuromuscular electrical stimulation (NMES) and sensory electrical stimulation (SES). NMES involves coordinated electrical stimulation of motor nerves and muscles to activate them with continuous short pulses of electrical current while SES involves stimulation of sensory nerves with electrical current resulting in sensations that vary from barely perceivable to highly unpleasant. Here, active cortical participation in rehabilitation procedures may be facilitated by driving the non-invasive electrotherapy with biosignals (electromyogram (EMG), electroencephalogram (EEG), electrooculogram (EOG)) that represent simultaneous active perception and volitional effort. To achieve this in a resource-poor setting, e.g., in low- and middle-income countries, we present a low-cost human-machine-interface (HMI) by leveraging recent advances in off-the-shelf video game sensor technology. In this paper, we discuss the open-source software interface that integrates low-cost off-the-shelf sensors for visual-auditory biofeedback with non-invasive electrotherapy to assist postural control during balance rehabilitation. We demonstrate the proof-of-concept on healthy volunteers.

A novel low-cost human-machine interface for interactive post-stroke balance rehabilitation system is presented in this article. The system integrates off-the-shelf low-cost sensors towards volitionally driven electrotherapy paradigm. The proof-of-concept software interface is demonstrated on healthy volunteers.

A stroke is caused when an artery carrying blood from heart to an area in the brain bursts or a clot obstructs the blood flow to brain thereby preventing ...

High-density Electroencephalographic Acquisition in a Rodent Model Using Low-cost and Open-source Resources

Advanced electroencephalographic analysis techniques requiring high spatial resolution, including electrical source imaging and measures of network connectivity, are applicable to an expanding variety of questions in neuroscience. Performing these kinds of analyses in a rodent model requires higher electrode density than traditional screw electrodes can accomplish. While higher-density electroencephalographic montages for rodents exist, they are of limited availability to most researchers, are not robust enough for repeated experiments over an extended period of time, or are limited to use in anesthetized rodents.1-3 A proposed low-cost method for constructing a durable, high-count, transcranial electrode array, consisting of bilaterally implantable headpieces is investigated as a means to perform advanced electroencephalogram analyses in mice or rats.

Procedures for headpiece fabrication and surgical implantation necessary to produce high signal to noise, low-impedance electroencephalographic and electromyographic signals are presented. While the methodology is useful in both rats and mice, this manuscript focuses on the more challenging implementation for the smaller mouse skull. Freely moving mice are only tethered to cables via a common adapter during recording. One version of this electrode system that includes 26 electroencephalographic channels and 4 electromyographic channels is described below.

Instructions for the low-cost construction and surgical implantation of a chronic transcranial high-density electroencephalographic montage into mice are provided. Signal recording, extraction, and processing techniques are also described.

Advanced electroencephalographic analysis techniques requiring high spatial resolution, including electrical source imaging and measures of network ...

The Power of Interstimulus Interval for the Assessment of Temporal Processing in Rodents

Temporal processing deficits have been implicated as a potential elemental dimension of higher-level cognitive processes, commonly observed in neurocognitive disorders. Despite the popularization of prepulse inhibition (PPI) in recent years, many current protocols promote using a percent of control measure, thereby precluding the assessment of temporal processing. The present study used cross-modal PPI and gap prepulse inhibition (gap-PPI) to demonstrate the benefits of employing a range of interstimulus intervals (ISIs) to delineate effects of sensory modality, psychostimulant exposure, and age. Assessment of sensory modality, psychostimulant exposure, and age reveals the utility of an approach varying the interstimulus interval (ISI) to establish the shape of the ISI function, including increases (sharper curve inflections) or decreases (flattening of the response amplitude curve) in startle amplitude. Additionally, shifts in peak response inhibition, suggestive of a differential sensitivity to the manipulation of ISI, are often revealed. Thus, the systematic manipulation of ISI affords a critical opportunity to evaluate temporal processing, which may reveal the underlying neural mechanisms involved in neurocognitive disorders.

Temporal processing, a preattentive process, may underlie deficits in higher-level cognitive processes, including attention, commonly observed in neurocognitive disorders. Using prepulse inhibition as an exemplar paradigm, we present a protocol for manipulating interstimulus interval (ISI) to establish the shape of the ISI function to provide an assessment of temporal processing.

Temporal processing deficits have been implicated as a potential elemental dimension of higher-level cognitive processes, commonly observed in ...

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.

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 ...

A Hydrophobic Tissue Clearing Method for Rat Brain Tissue

Hydrophobic tissue clearing methods are easily adjustable, fast, and low-cost procedures that allows for the study of a molecule of interest in unaltered tissue samples. Traditional immunolabeling procedures require cutting the sample into thin sections, which restricts the ability to label and examine intact structures. However, if brain tissue can remain intact during processing, structures and circuits can remain intact for the analysis. Previously established clearing methods take significant time to completely clear the tissue, and the harsh chemicals can often damage sensitive antibodies. The iDISCO method quickly and completely clears tissue, is compatible with many antibodies, and requires no special lab equipment. This technique was initially validated for the use in mice tissue, but the current protocol adapts this method to image hemispheres of control and transgenic rat brains. In addition to this, the present protocol also makes several adjustments to preexisting protocol to provide clearer images with less background staining. Antibodies for Iba-1 and tyrosine hydroxylase were validated in the HIV-1 transgenic rat and in F344/N control rats using the present hydrophobic tissue clearing method. The brain is an interwoven network, where structures work together more often than separately of one another. Analyzing the brain as a whole system as opposed to a combination of individual pieces is the greatest benefit of this whole brain clearing method.

Here we present a hydrophobic tissue clearing method that allows for the viewing of target molecules as part of intact brain structures. This technique has now been validated for F344/N control and HIV-1 transgenic rats of both sexes.

Hydrophobic tissue clearing methods are easily adjustable, fast, and low-cost procedures that allows for the study of a molecule of interest in unaltered ...