Funding for this research was provided by The Garnett Passes and Rodney Williams Memorial Foundation and La Trobe University.

<ol>
	<li>Mauger, S. J., Shivdasani, M. N., Rathbone, G. D., Argent, R. E., Paolini, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+in+vivo+investigation+of+first+spike+latencies+in+the+inferior+colliculus+in+response+to+multichannel+penetrating+auditory+brainstem+implant+stimulation.">An in vivo investigation of first spike latencies in the inferior colliculus in response to multichannel penetrating auditory brainstem implant stimulation.</a> Journal of Neural Engineering. 7, 036004-036004 (2010).</li><li>Shivdasani, M. N., Mauger, S. J., Argent, R. E., Rathbone, G. D., Paolini, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inferior+colliculus+responses+to+dual-site+intralamina+stimulation+in+the+ventral+cochlear+nucleus.">Inferior colliculus responses to dual-site intralamina stimulation in the ventral cochlear nucleus.</a> J. Comp. Neurol. 518, 4226-4242 (2010).</li><li>Shivdasani, M. N., Mauger, S. J., Rathbone, G. D., Paolini, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inferior+colliculus+responses+to+multichannel+microstimulation+of+the+ventral+cochlear+nucleus:+Implications+for+auditory+brain+stem+implants.">Inferior colliculus responses to multichannel microstimulation of the ventral cochlear nucleus: Implications for auditory brain stem implants.</a> Journal of Neurophysiology. 99, 1-13 (2007).</li><li>Lenarz, M., Lim, H. H., Patrick, J. F., Anderson, D. J., Lenarz, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophysiological+Validation+of+a+Human+Prototype+Auditory+Midbrain+Implant+in+a+Guinea+Pig+Model.">Electrophysiological Validation of a Human Prototype Auditory Midbrain Implant in a Guinea Pig Model.</a> JARO. 7, 383-398 (2006).</li><li>Lim, H. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Auditory+Cortical+Responses+to+Electrical+Stimulation+of+the+Inferior+Colliculus:+Implications+for+an+Auditory+Midbrain+Implant.">Auditory Cortical Responses to Electrical Stimulation of the Inferior Colliculus: Implications for an Auditory Midbrain Implant.</a> Journal of Neurophysiology. 96, 975-988 (2006).</li><li>Otto, K. J., Rousche, P. J., Kipke, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microstimulation+in+auditory+cortex+provides+a+substrate+for+detailed+behaviors.">Microstimulation in auditory cortex provides a substrate for detailed behaviors.</a> Hearing research. 210, 112-117 (2005).</li><li>Gai, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+Tones+in+Reproducible+Noise+Maskers+by+Rabbits+and+Comparison+to+Detection+by+Humans.">Detection of Tones in Reproducible Noise Maskers by Rabbits and Comparison to Detection by Humans.</a> JARO. 8, 522-538 (2007).</li><li>Miller, C. A., Woodruff, K. E., Pfingst, B. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+responses+from+guinea+pigs+with+cochlear+implants.+I.+Electrophysiological+and+psychophysical+measures.">Functional responses from guinea pigs with cochlear implants. I. Electrophysiological and psychophysical measures.</a> Hearing Research. 92, 85-99 (1995).</li><li>Beitel, R. E., Vollmer, M., Snyder, R. L., Schreiner, C. E., Leake, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavioral+and+neurophysiological+thresholds+for+electrical+cochlear+stimulation+in+the+deaf+cat.">Behavioral and neurophysiological thresholds for electrical cochlear stimulation in the deaf cat.</a> Audiology &#38; Neurotology. 5, 31-38 (2000).</li><li>Beitel, R. E., Snyder, R. L., Schreiner, C. E., Raggio, M. W., Leake, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrical+cochlear+stimulation+in+the+deaf+cat:+comparisons+between+psychophysical+and+central+auditory+neuronal+thresholds.">Electrical cochlear stimulation in the deaf cat: comparisons between psychophysical and central auditory neuronal thresholds.</a> J. Neurophysiol. 83, 2145-2162 (2000).</li><li>Vollmer, M., Beitel, R. E., Snyder, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Auditory+detection+and+discrimination+in+deaf+cats:+Psychophysical+and+neural+thresholds+for+intracochlear+electrical+signals.">Auditory detection and discrimination in deaf cats: Psychophysical and neural thresholds for intracochlear electrical signals.</a> Journal of Neurophysiology. 86, 2330-2343 (2001).</li><li>LeDoux, J. E., Cicchetti, P., Xagoraris, A., Romanski, L. 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+lateral+amygdaloid+nucleus:+sensory+interface+of+the+amygdala+in+fear+conditioning.">The lateral amygdaloid nucleus: sensory interface of the amygdala in fear conditioning.</a> Journal of Neuroscience. 10, 1062-1069 (1990).</li><li>McCreery, D. B., Yuen, T. G., Agnew, W. F., Bullara, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+characterization+of+the+effects+on+neuronal+excitability+due+to+prolonged+microstimulation+with+chronically+implanted+microelectrodes.">A characterization of the effects on neuronal excitability due to prolonged microstimulation with chronically implanted microelectrodes.</a> IEEE Transactions on Biomedical Engineering. 44, 931-939 (1997).</li><li>Liu, X., McPhee, G., Seldon, H. L., Clark, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histological+and+physiological+effects+of+the+central+auditory+prosthesis:+surface+versus+penetrating+electrodes.">Histological and physiological effects of the central auditory prosthesis: surface versus penetrating electrodes.</a> Hearing Research. 114, 264-274 (1997).</li><li>Polikov, V. S., Tresco, P. A., Reichert, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Response+of+brain+tissue+to+chronically+implanted+neural+electrodes.">Response of brain tissue to chronically implanted neural electrodes.</a> Journal of Neuroscience Methods. 148, 1-18 (2005).</li></ol>

Dr. Antonio Paolini is a Director of Bioengenesis Technologies Pty Ltd who are the Australian representatives for Tucker Davis Technologies and the Australian distributor of NeuroNexus Probes.

The techniques described here provide a means for testing a range of discrimination tasks rapidly, with short training times and allowing for substantial automation, thus minimising researcher time required for data processing. Using data obtained from the implanted ECG, calculation of heart-rate (HR) change at times of stimulus presentation can be automated. Errors in ECG processing (such as missing a single heart-beat) can be easily identified as they produce spuriously large changes in apparent heart-rate for a very brief period, and such easy error detection minimises the requirement for time-intensive manual data review.
The technique described for the implantation of the ECG telemetry device consistently produces recordings with little or no interference (see Figures 1 &amp; 2). However, relatively small variations in the placement of the lead in the mediastinum region can lead to interference from nearby muscles and particular respiration-associated noise. Verifying correct placement of the leads during the implantation process, particularly after suturing leads in place, allows fine adjustments of lead placement to minimise interference.
The process of brain implantation into the cochlear nucleus is difficult. By using serial presentations of band-pass filtered noise of several frequency ranges, rather than broadband noise as has previously been used3, responses of cell populations at electrode sites can be rapidly reviewed in terms of frequency-response rather than the less-specific noise response. The ability to rapidly identify when a sufficient range of frequency-tuned cell populations are accessible is critical in research when electrical stimulus discrimination is being tested. If insufficient frequency-distribution is obtained, then comparison between stimulation of distinct frequency areas is simply not possible. Nonetheless, the longer process of frequency-amplitude response mapping remains critical to providing detail regarding the broadness of tuning of cell populations and should be performed prior to fixing the electrode in place.
In behavioral training and testing, the inclusion of a non-stimulus (or silent) period between trials, prior to stimulus onset, allows the response to initial stimulus onset to be examined. When using electrical neural stimulation, a response to stimulus onset provides evidence for the simple detection of the stimulus. Thus, when there is no stimulus onset response, but a response when the second alternation stimulus is introduced, it can be deduced that only the latter stimulus can be detected. Without inclusion of such a silent period, there would be no way to determine that the former stimulus was not detected.
A limitation in this behavioral testing technique is that the absence of foot-shocks delivered after the presentation of neural stimulation may lead to the development of specificity in conditioned fear, such that fear is only expressed when acoustic stimulus is presented. That is, the animal may learn that the particular sensory effects of neural stimulation are associated with not receiving a foot shock. In contrast, delivering foot-shocks after the presentation of neural stimulation could lead to a conditioned fear of the neural stimulus itself, distinct from any sound-like properties the neural stimulus may have. Results obtained when excluding a foot-shock after neural stimulation certainly provide stronger evidence for neural stimulus being sound-like than those using a foot-shock after neural stimulation, as in the former case conditioned fear has generalised from acoustic to neural stimulation. Nonetheless, the risk of developing specificity to acoustic rather than neural stimulation is present. The use of band-pass filtered noise as acoustic stimulus rather than pure tone bursts, the former of which may better reflect the perceptual experience of neural stimulation, could reduce the risk of such specificity developing. However, such a procedure introduces the additional variable of band-width of the filter, which will affect discrimination tasks.
An additional limitation associated with any chronic neural implantation, such as required for behavioral testing, is the change in function of the electrode or the associated neural tissue over time. Stimulation of neural tissue can lead to both temporary suppression of neural activity13 and tissue changes such as cell loss14 and direct brain tissue response to the electrode.15
The described approach to implantation and behavioral testing provides a means to test both detection and discrimination of acoustic and electrical stimuli with brief training, and the ability to exert control of trial frequency. The technique of using change in HR as a measure of conditioned fear may be applicable to not only auditory testing, but more generally to any sensory testing in which stimuli can be presented for any discrete period, and in which detection of sensory discrimination is desired.

<table border="1">
<tbody>
 <tr>
 <td>Name</td>
 <td>Company</td>
 <td>Model</td>
 <td>Comments </td>
 </tr>
 <tr>
 <td>PowerLab</td>
 <td>ADInstruments</td>
 <td>ML880</td>
 <td>Records data received from the implanted TR40 transmitter</td>
 </tr>
 <tr>
 <td>SmartCtrl controller card</td>
 <td>Med Associates</td>
 <td>DIG-716B</td>
 <td>Controls the behavioral test chamber, including foot shock</td>
 </tr>
 <tr>
 <td>Modular behavioral test chamber</td>
 <td>Med Associates</td>
 <td>ENV-009</td>
 <td>Test chamber size: 30.5cm x 39.4cm</td>
 </tr>
 <tr>
 <td>Aversive stimulus generator</td>
 <td>Med Associates</td>
 <td>ENV-410B</td>
 <td>Delivers aversive foot-shock to metal-bar floor of the cage through a Solid State Scrambler unit</td>
 </tr>
 <tr>
 <td>Programmable Attenuator</td>
 <td>TDT</td>
 <td>PA5</td>
 <td>Controls the amplitude of delivered acoustic signal to ensure stable amplitude across frequencies</td>
 </tr>
 <tr>
 <td>Electrostatic speaker driver</td>
 <td>TDT</td>
 <td>ED1</td>
 <td>Drives the electrostatic speakers (EC1, ES1)</td>
 </tr>
 <tr>
 <td>Free-field electrostatic speaker</td>
 <td>TDT</td>
 <td>ES1</td>
 <td>Sounds are presented using this speaker in the behavioral test chamber</td>
 </tr>
 <tr>
 <td>Coupled electrostatic speaker</td>
 <td>TDT</td>
 <td>EC1</td>
 <td>Sounds are presented using this speaker during neural implant surgery</td>
 </tr>
 <tr>
 <td>Stimulator Base Station</td>
 <td>TDT</td>
 <td>RX7</td>
 <td>Controls delivery of electrical neural stimulation (used with MS16)</td>
 </tr>
 <tr>
 <td>Microstimulator</td>
 <td>TDT</td>
 <td>MS16</td>
 <td>Delivers multichannel electrical neural stimulation</td>
 </tr>
 <tr>
 <td>Processing base station</td>
 <td>TDT</td>
 <td>RZ2</td>
 <td>Records neural activity during brain implantation (using PZ2 preamplifier)</td>
 </tr>
 <tr>
 <td>Preamplifier</td>
 <td>TDT</td>
 <td>PZ2-256</td>
 <td>256-channel high impedance preamplifier</td>
 </tr>
 <tr>
 <td>Telemetry device receiver</td>
 <td>Telemetry Research</td>
 <td>TR162</td>
 <td>Receives digital signal from TR40, and converts to amplified analogue output</td>
 </tr>
 <tr>
 <td>Implantable electrocardiogram telemetry device</td>
 <td>Telemetry Research</td>
 <td>TR40</td>
 <td>The implanted transmitter device, sampling at 2kHz</td>
 </tr>
 <tr>
 <td>Multifunction Processor</td>
 <td>Tucker Davis Technologies (TDT)</td>
 <td>RX6</td>
 <td>Used to generate acoustic stimuli</td>
 </tr>
 <tr>
 <td>Vertex Castavaria</td>
 <td>Vertex Dental</td>
 <td>&nbsp;</td>
 <td>Dental acrylic used to fix the electrode in place</td>
 </tr>
 <tr>
 <td>Kwik-Sil Adhesive, 
 low viscosity</td>
 <td>World Precision Instruments</td>
 <td>&nbsp;</td>
 <td>Silicon elastomer used to coat the electrode shanks</td>
 </tr>
 <tr>
 <td>Multichannel electrode</td>
 <td>NeuroNexus</td>
 <td>a2x16-10mm 
 100-500-413 </td>
 <td>The 2-shank 32-channel extracellular electrode array used for implantation. The electrode sites have been activated to produce a coating of iridium oxide in preparation for stimulation. </td>
 </tr>
</tbody>
</table>

behavioral determination of stimulus pair discrimination of auditory acoustic and electrical stimuli using a classical conditioning and heart-rate approach

Acute animal preparations have been used in research prospectively investigating electrode designs and stimulation techniques for integration into neural auditory prostheses, such as auditory brainstem implants1-3 and auditory midbrain implants4,5. While acute experiments can give initial insight to the effectiveness of the implant, testing the chronically implanted and awake animals provides the advantage of examining the psychophysical properties of the sensations induced using implanted devices6,7.
Several techniques such as reward-based operant conditioning6-8, conditioned avoidance9-11, or classical fear conditioning12 have been used to provide behavioral confirmation of detection of a relevant stimulus attribute. Selection of a technique involves balancing aspects including time efficiency (often poor in reward-based approaches), the ability to test a plurality of stimulus attributes simultaneously (limited in conditioned avoidance), and measure reliability of repeated stimuli (a potential constraint when physiological measures are employed).
Here, a classical fear conditioning behavioral method is presented which may be used to simultaneously test both detection of a stimulus, and discrimination between two stimuli. Heart-rate is used as a measure of fear response, which reduces or eliminates the requirement for time-consuming video coding for freeze behaviour or other such measures (although such measures could be included to provide convergent evidence). Animals were conditioned using these techniques in three 2-hour conditioning sessions, each providing 48 stimulus trials. Subsequent 48-trial testing sessions were then used to test for detection of each stimulus in presented pairs, and test discrimination between the member stimuli of each pair. 
This behavioral method is presented in the context of its utilisation in auditory prosthetic research. The implantation of electrocardiogram telemetry devices is shown. Subsequent implantation of brain electrodes into the Cochlear Nucleus, guided by the monitoring of neural responses to acoustic stimuli, and the fixation of the electrode into place for chronic use is likewise shown.

Watch this Scientific Journal Video about Behavioral Determination of Stimulus Pair Discrimination of Auditory Acoustic and Electrical Stimuli Using a Classical Conditioning and Heart-rate Approach at

Behavioral Determination of Stimulus Pair Discrimination of Auditory Acoustic and Electrical Stimuli Using a Classical Conditioning and Heart-rate Approach

The application of a classical fear conditioning behavioral paradigm for auditory prosthetic research in rats is described. This paradigm provides a mechanism for identifying both detection of, and discrimination between, distinct acoustic and electrical stimuli using heart-rate as an outcome measure.

Acute animal preparations have been used in research prospectively investigating electrode designs and stimulation techniques for integration into neural ...

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14.4K Views.  La Trobe University. The application of a classical fear conditioning behavioral paradigm for auditory prosthetic research in rats is described. This paradigm provides a mechanism for identifying both detection of, and discrimination between, distinct acoustic and electrical stimuli using heart-rate as an outcome measure.

Video: Behavioral Determination of Stimulus Pair Discrimination of Auditory Acoustic and Electrical Stimuli Using a Classical Conditioning and Heart-rate Approach

Behavioural Pharmacology in Classical Conditioning of the Proboscis Extension Response in Honeybees (Apis mellifera)

Honeybees (Apis mellifera) are well known for their communication and orientation skills and for their impressive learning capability1,2. Because the survival of a honeybee colony depends on the exploitation of food sources, forager bees learn and memorize variable flower sites as well as their profitability. Forager bees can be easily trained in natural settings where they forage at a feeding site and learn the related signals such as odor or color. Appetitive associative learning can also be studied under controlled conditions in the laboratory by conditioning the proboscis extension response (PER) of individually harnessed honeybees3,4. This learning paradigm enables the study of the neuronal and molecular mechanisms that underlie learning and memory formation in a simple and highly reliable way5-12. A behavioral pharmacology approach is used to study molecular mechanisms. Drugs are injected systemically to interfere with the function of specific molecules during or after learning and memory formation13-16.
Here we demonstrate how to train harnessed honeybees in PER conditioning and how to apply drugs systemically by injection into the bee flight muscle.

Behavioural Pharmacology in Classical Conditioning of the Proboscis Extension Response in Honeybees Apis mellifera

We demonstrate how to implement a behavioral pharmacology method in an appetitive olfactory conditioning paradigm in honeybees (Apis mellifera) by systemic application of drugs. This method allows investigation of the mechanisms underlying learning and memory formation in a simple and reliable way.

Honeybees (Apis mellifera) are well known for their communication and orientation skills and for their impressive learning capability1,2. Because the ...

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 ...

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

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

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

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

High Resolution Quantitative Synaptic Proteome Profiling of Mouse Brain Regions After Auditory Discrimination Learning

The molecular synaptic mechanisms underlying auditory learning and memory remain largely unknown. Here, the workflow of a proteomic study on auditory discrimination learning in mice is described. In this learning paradigm, mice are trained in a shuttle box Go/NoGo-task to discriminate between rising and falling frequency-modulated tones in order to avoid a mild electric foot-shock. The protocol involves the enrichment of synaptosomes from four brain areas, namely the auditory cortex, frontal cortex, hippocampus, and striatum, at different stages of training. Synaptic protein expression patterns obtained from trained mice are compared to na&#239;ve controls using a proteomic approach. To achieve sufficient analytical depth, samples are fractionated in three different ways prior to mass spectrometry, namely 1D SDS-PAGE/in-gel digestion, in-solution digestion and phospho-peptide enrichment. 
High-resolution proteomic analysis on a mass spectrometer and label-free quantification are used to examine synaptic protein profiles in phospho-peptide-depleted and phospho-peptide-enriched fractions of synaptosomal protein samples. A commercial software package is utilized to reveal proteins and phospho-peptides with significantly regulated relative synaptic abundance levels (trained/na&#239;ve controls). Common and differential regulation modes for the synaptic proteome in the investigated brain regions of mice after training were observed. Subsequently, meta-analyses utilizing several databases are employed to identify underlying cellular functions and biological pathways.

The identification of molecules and pathways controlling synaptic plasticity and memory is still a major challenge in neuroscience. Here, a workflow is described addressing the relative quantification of synaptic proteins supposedly involved in the molecular reorganization of synapses during learning and memory consolidation in an auditory learning paradigm.

The molecular synaptic mechanisms underlying auditory learning and memory remain largely unknown. Here, the workflow of a proteomic study on auditory ...

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

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

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

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

Drosophila Courtship Conditioning As a Measure of Learning and Memory

Many insights into the molecular mechanisms underlying learning and memory have been elucidated through the use of simple behavioral assays in model organisms such as the fruit fly, Drosophila melanogaster. Drosophila is useful for understanding the basic neurobiology underlying cognitive deficits resulting from mutations in genes associated with human cognitive disorders, such as intellectual disability (ID) and autism. This work describes a methodology for testing learning and memory using a classic paradigm in Drosophila known as courtship conditioning. Male flies court females using a distinct pattern of easily recognizable behaviors. Premated females are not receptive to mating and will reject the male&#39;s copulation attempts. In response to this rejection, male flies reduce their courtship behavior. This learned reduction in courtship behavior is measured over time, serving as an indicator of learning and memory. The basic numerical output of this assay is the courtship index (CI), which is defined as the percentage of time that a male spends courting during a 10 min interval. The learning index (LI) is the relative reduction of CI in flies that have been exposed to a premated female compared to na&#239;ve flies with no previous social encounters. For the statistical comparison of LIs between genotypes, a randomization test with bootstrapping is used. To illustrate how the assay can be used to address the role of a gene relating to learning and memory, the pan-neuronal knockdown of Dihydroxyacetone phosphate acyltransferase (Dhap-at) was characterized here. The human ortholog of Dhap-at, glyceronephosphate O-acyltransferase (GNPT), is involved in rhizomelic chondrodysplasia punctata type 2, an autosomal-recessive syndrome characterized by severe ID. Using the courtship conditioning assay, it was determined that Dhap-at is required for long-term memory, but not for short-term memory. This result serves as a basis for further investigation of the underlying molecular mechanisms.

Drosophila Courtship Conditioning As a Measure of Learning and Memory

This protocol describes a Drosophila learning and memory assay called courtship conditioning. This classic assay is based on a reduction of male courtship behavior after sexual rejection by a non-receptive premated female. This natural form of behavioral plasticity can be used to test learning, short-term memory, and long-term memory.

Many insights into the molecular mechanisms underlying learning and memory have been elucidated through the use of simple behavioral assays in model ...

Quantifying Infra-slow Dynamics of Spectral Power and Heart Rate in Sleeping Mice

Three vigilance states dominate mammalian life: wakefulness, non-rapid eye movement (non-REM) sleep, and REM sleep. As more neural correlates of behavior are identified in freely moving animals, this three-fold subdivision becomes too simplistic. During wakefulness, ensembles of global and local cortical activities, together with peripheral parameters such as pupillary diameter and sympathovagal balance, define various degrees of arousal. It remains unclear the extent to which sleep also forms a continuum of brain states-within which the degree of resilience to sensory stimuli and arousability, and perhaps other sleep functions, vary gradually-and how peripheral physiological states co-vary. Research advancing the methods to monitor multiple parameters during sleep, as well as attributing to constellations of these functional attributes, is central to refining our understanding of sleep as a multifunctional process during which many beneficial effects must be executed. Identifying novel parameters characterizing sleep states will open opportunities for novel diagnostic avenues in sleep disorders.
We present a procedure to describe dynamic variations of mouse non-REM sleep states via the combined monitoring and analysis of electroencephalogram (EEG)/electrocorticogram (ECoG), electromyogram (EMG), and electrocardiogram (ECG) signals using standard polysomnographic recording techniques. Using this approach, we found that mouse non-REM sleep is organized into cycles of coordinated neural and cardiac oscillations that generate successive 25-s intervals of high and low fragility to external stimuli. Therefore, central and autonomic nervous systems are coordinated to form behaviorally distinct sleep states during consolidated non-REM sleep. We present surgical manipulations for polysomnographic (i.e., EEG/EMG combined with ECG) monitoring to track these cycles in the freely sleeping mouse, the analysis to quantify their dynamics, and the acoustic stimulation protocols to assess their role in the likelihood of waking up. Our approach has already been extended to human sleep and promises to unravel common organizing principles of non-REM sleep states in mammals.

Here, we present experimental and analytical procedures to describe the temporal dynamics of the neural and cardiac variables of non-REM sleep in mice, which modulate sleep responsiveness to acoustic stimuli.

Three vigilance states dominate mammalian life: wakefulness, non-rapid eye movement (non-REM) sleep, and REM sleep. As more neural correlates of behavior ...

Desensitization and Recovery of Crayfish Photoreceptors Upon Delivery of a Light Stimulus

A method to study desensitization and recovery of crayfish photoreceptors is presented. We performed intracellular electrical recordings of photoreceptor cells in isolated eyestalks using the discontinuous single electrode-switched voltage-clamp configuration. First, with a razor blade we made an opening in the dorsal cornea to get access to the retina. Thereafter, we inserted a glass electrode through the opening, and penetrated a cell as reported by the recording of a negative potential. Membrane potential was clamped at the photoreceptor's resting potential and a light-pulse was applied to activate currents. Finally, the two light-flash protocol was employed to measure current desensitization and recovery. The first light-flash triggers, after a lag period, the transduction ionic current, which after reaching a peak amplitude decays towards a desensitized state; the second flash, applied at varying time intervals, assesses the state of the light-activated conductance. To characterize the light-elicited current, three parameters were measured: 1) latency (the time elapsed between light flash delivery and the moment in which current achieves 10% of its maximum value); 2) peak current; and 3) desensitization time constant (exponential time constant of the current decay phase). All parameters are affected by the first pulse.
To quantify recovery from desensitization, the ratio p2/p1 was employed versus time between pulses. p1 is the peak current evoked by the first light-pulse, and p2 is the peak current evoked by the second pulse. These data were fitted to a sum of exponential functions. Finally, these measurements were carried out as function of circadian time.

A protocol for the study of desensitization and sensitivity recovery of crayfish photoreceptors as a function of circadian time is presented.

A method to study desensitization and recovery of crayfish photoreceptors is presented. We performed intracellular electrical recordings of photoreceptor ...

An Objective and Reproducible Test of Olfactory Learning and Discrimination in Mice

Olfaction is the predominant sensory modality in mice and influences many important behaviors, including foraging, predator detection, mating, and parenting. Importantly, mice can be trained to associate novel odors with specific behavioral responses to provide insight into olfactory circuit function. This protocol details the procedure for training mice on a Go/No-Go operant learning task. In this approach, mice are trained on hundreds of automated trials daily for 2&#8211;4 weeks and can then be tested on novel Go/No-Go odor pairs to assess olfactory discrimination, or be used for studies on how odor learning alters the structure or function of the olfactory circuit. Additionally, the mouse olfactory bulb (OB) features ongoing integration of adult-born neurons. Interestingly, olfactory learning increases both the survival and synaptic connections of these adult-born neurons. Therefore, this protocol can be combined with other biochemical, electrophysiological, and imaging techniques to study learning and activity-dependent factors that mediate neuronal survival and plasticity.

Here, we train mice on an associative learning task to test odor discrimination. This protocol also allows for studies on learning-induced structural changes in the brain.

Olfaction is the predominant sensory modality in mice and influences many important behaviors, including foraging, predator detection, mating, and ...

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 ...