Supported by the National Institutes of Health (NIDCD, P30-DC010754, T32-DC009401).

1.	Pre/Post-training Recordings
<ol>
<li>Begin ultrasonic monitoring with Avisoft Recorder using the configuration shown in Figure 2.</li>
<li>Ultrasonic vocalizations can be monitored either visually by watching the real-time spectrogram, or aurally with headphones plugged into the ultrasound recording interface.</li>
<li>Place a sexually receptive (in estrus) female rat into the home cage of the subject male rat. Signs of estrus include lordosis, ear wiggling, and rapid darting. Females in estrus elicit more vocalizations from males than non-sexually receptive females.</li>
<li>After the male has shown interest in the female (e.g., sniffing, chasing, and/or mounting), remove the female and record the male's vocalizations for 1-2 minutes.</li>
<li>If the male does not vocalize immediately, briefly return the female rat to the male's home cage to stimulate vocalization.</li>
</ol>
2.	Vocal Training 
<ol>
<li>Begin ultrasonic monitoring with Avisoft Recorder using the same configuration shown in Figure 2.</li>
<li>Place the male rat in his home cage under the microphone.</li>
<li>Put a female rat into the male's home cage. As stated previously, the female rat should be in estrus to maximize the vocalization response from the male.</li>
<li>Once the male has shown interest in the female remove the female.</li>
<li>Immediately after the male produces the target vocalization, reward simultaneously with a pen click and a brief presentation of the water bottle or food treat. Initially, any 50-kHz frequency modulated (Figure 5B) call is rewarded. As training progresses only increasingly complex strings of calls, such as strings of 5 to 10 calls in rapid succession, are reinforced.</li>
<li>Continue the training session until you have given 30 reinforcements. This typically takes between 5 &#x2013; 10 minutes.</li>
</ol>
3.	Preparing for Acoustic Analysis
<ol>
<li>Always archive the original sound files and make edits and measurements on a working copy, as the analysis will require filtering and erasing noise from each file.</li> 
<li>Using the batch mode of Avisoft SASLab Pro, high pass filter all the sound files to be analyzed at 25 kHz to remove unwanted noise below the ultrasonic vocalizations.</li>
<li>In order to use the automatic detection feature of SASLab Pro, the noise threshold must first be determined. To do this, open a sound file and create a spectrogram using the settings shown in Figure 3.</li>
<li>The Automatic Parameter Measurements display should be setup according to the settings shown in Figure 4.</li>
<li>Find an area of the spectrogram where there are no calls or noises; this is the background noise.</li>
<li>Using the "automatic (single threshold)" option, set the threshold in the Element separation section of the Automatic Parameter Measurements setup dialog box to just above the background noise by adjusting the black line in the power spectrum window above the spectrogram. In Figure 4, the threshold has been set to -55 dB.</li>
<li>At the bottom of the dialog box, set the "Reject if peak amplitude is less than" value to 1 dB below the threshold found in the previous step. These two values should now be used for all sound files analyzed, assuming all recording settings were the same.</li>
</ol>
4.	Vocalization Identification
<ol>
<li>To label the vocalizations using the automatic threshold set above, first create a spectrogram with the above settings.</li>
<li>Manually erase any noise that meets the following criteria: 1) has been wrongly identified as a vocalization, 2) is affecting the discrimination of the beginning and/or end of the vocalization, and/or 3) has been wrongly identified as the highest or lowest frequency in the vocalization (indicated by the upper and lower red lines, respectively). Alternatively, the start/stop times of the vocalization labels can be adjusted manually after creating permanent labels in step 4.5 below. Noise affecting the high and low frequency measurements, however, must be manually erased.</li>
<li>In some cases a vocalization will not be able to be separated from the surrounding noises, such as the sound of bedding during locomotion. In these cases, the call cannot be accurately analyzed and should be erased.</li>
<li>Once all vocalizations have been identified, permanently erase the deleted sections of the spectrogram from the sound file by selecting "Remove erased spectrogram sections from waveform&#x2026;" from the Tools drop-down menu. If this command is not executed before the spectrogram is closed, all erasing work will be lost.</li>
<li>Create permanent labels in the sound file by clicking the 'edit' button in the Element Separation section of the Automatic Parameter Measurements setup window.</li>
<li>Visually and aurally review each labeled vocalization to determine its category: simple, frequency modulated (FM), or harmonic. Assign the appropriate label using the pre-defined text-modules. Examples of each vocalization category are shown in Figure 5.</li>
<li>Close the spectrogram, save the sound file, and continue with the next sound file to be analyzed.</li>
</ol>
5.	Acoustic Measurements
<ol>
<li>After vocalizations in all the sound files have been identified, use the batch processing tool (Tools: Batch Processing) to automatically measure the acoustic parameters of all the files.</li>
<li>In the Batch processing dialogue box, select "Automatic Parameter Measurements" from the drop-down menu.</li>
<li>Select the "process all files in the selected folder" checkbox then click the "folder" button and select the folder where the sound files are saved.</li>
<li>Click "Start" and each file will be automatically analyzed according to the settings last used in the Automatic Parameter Measurement setup dialogue box.</li> 
<li>The measurements will be saved in a text file that can be defined using the DDE Parameters/Log File Setting command. This text file can then be imported into a program for statistical analysis.</li>
</ol>
6. Representative Results: 
Ultrasonic vocalizations of rats are affected by physiological changes associated with aging and 6-OHDA Parkinson disease models. 13 Typically, we see a reduction in quality of the ultrasonic vocalizations characterized by a reduction of the following quantitative acoustic parameters: bandwidth, peak frequency, duration, and intensity. Figure 6 shows the frequency modulated vocalizations of a rat in the PD model at three different time points: baseline, after induction of PD, and after vocalization training. In the PD condition, the call shows a reduction in bandwidth, duration, and intensity. Additionally, the frequency modulation has become irregular.
In addition to degradation of specific acoustic parameters, we also observe less overall complexity in the types of vocalizations produced in the aged and parkinsonism rats. For example, after induction of PD a rat produces a greater number of simple calls and fewer frequency modulated calls. Following vocalization training, many acoustic parameters, such as duration and intensity, approach baseline levels and the number of complex calls increases (Figure 6). Aged rats manifest similar degradation of acoustic parameters in their ultrasonic vocalizations. We are currently investigating the effects of vocal training in aged rats. 
<img src="/files/ftp_upload/2835/2835fig1.jpg" alt="Figure 1"/> 
Figure 1. Number of vocalizations per session (target met by all rats each week) and vocalization rate (mean and standard error shown) both increase over a 6-week training period in a group of both young (9 mo) and old (32 mo) Fischer 344/Brown Norway F1 rats.
<img src="/files/ftp_upload/2835/2835fig2.jpg" alt="Figure 2"/> 
Figure 2. Representative vocalizations of each of the three categories of 50 kHz vocalizations; (A) simple, (B) frequency-modulated, and (C) harmonic.
<img src="/files/ftp_upload/2835/2835fig3.jpg" alt="Figure 3"/> 
Figure 3. Representative frequency-modulated vocalizations from a single rat at (A) baseline, (B) post-PD induction, and (C) after vocalization training.

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Experiments on animals were performed in accordance with the guidelines and regulations set forth by the Public Health Service Policy on Humane Care and Use of Laboratory Animals, the NIH guide for care and use of laboratory animals, and the animal welfare act. The animal use protocol was approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Wisconsin-Madison School of Medicine and Public Health.

Ultrasonic vocalizations in rats appear to be vulnerable to aging and disease processes, such as Parkinson disease. These ultrasonic vocalizations provide a behavioral model of vocal function, including deficits that accompany physiological changes due to disease and aging. In addition, acoustic changes in ultrasonic vocalizations are easily measured and quantified. Further, ultrasonic vocalizations can be modified through behavioral interventions. Therefore, rat ultrasonic vocalizations provide a useful model to study the impact of disease, aging, and therapeutic interventions on both behavior and underlying neurobiological mechanisms.
Our method of vocal training uses a conditioned stimulus (a click) associated with a positive reinforcement (a water or food treat reward). The click followed by the reward is used to reinforce vocalizations of increasing complexity. We define complex vocalizations as those containing a "trill" (frequency modulated) component (Figure 5B) versus those that are "flat" and maintain a single frequency (Figure 5A). This frequency modulated component is easily identified by its sinusoidal appearance. While 50-kHz vocalizations may be entirely sinusoidal (Figure 5B), it is common to encounter more complex 50-kHz vocalizations that consist partly of this frequency modulated component as well as either large jumps in frequency or a brief flat component. These more complex vocalizations may have short breaks of less than 10 ms between components but are considered a single vocalization. Although further detailed sub-classification of these vocalizations is possible offline,14 we classify all 50-kHz vocalizations containing a frequency modulated component as complex to reduce the need for online subjective classification and reduce reinforcement variability. In your own lab it is important to choose how data is classified and analyzed according a study design that is appropriate for the purpose of the study.
There are many factors to consider when using this protocol. First of all, despite genetic homogeneity and procedural consistency, rats have a variable response to training. Some rats naturally vocalize more than others,5 or are motivated by exposure to a female more than by a food or water reward. One way we account for this variability is by examining the change in acoustic parameters from individual rats' baseline vocalizations to their post-lesion and post-training vocalizations. Although not discussed in this protocol, we also have behavioral assays to quantify interest in the female, such as latency to mount. Additionally, depending on the age and activity level of an individual rat, a wire top may be needed on the home cage during training to prevent escape. Despite individual variability, we have successfully used this protocol on a variety of strains and ages to improve vocalizations relative to each individual rat's baseline.
Rats are sensitive to seemingly small changes in the environment, such as scents, temperature, and personnel. An effort should be made to avoid scented personal care products, such as lotions, hair products, and perfumes. Before attempting training, an individual must be comfortable interacting with and handling rats. Furthermore, any inconsistencies in personnel or environment may affect the rats' vocalization response.
Because rats are nocturnal, they should be kept on a reversed light cycle to allow for training to take place during the dark portion of their cycle when they are most active. We illuminate our training room with red lights. Additionally, if a water reward is used during training, rats should have restricted access to water. Our rats are water restricted for 21 hours prior to the training session. Three hours of water exposure was determined by our Institutional Animal Care and Use Committee to be the least restrictive time period that allowed water to be useful as reinforcement in our behavioral studies without presenting a substantial compromise to animal health or well being. Rats are given access to water at random times (10 minutes to 30 minutes) after training and subsequently have free access to water for three hours during the dark portion of their light cycle. Rats should be inspected every day for signs of dehydration and weighed every other day to ensure there is no significant (greater than 10%) weight loss. A food restriction is not necessary if using a food treat as a reward. Treats specific to rats are commercially available, but we have found a small piece of a sugary cereal to be an effective reward.
As mentioned above, female rats in estrus elicit a higher vocalization response from males than non-sexually receptive females. By maintaining a large number of females in the colony, there is a high likelihood that at least one female will be in estrus on any given day. However, estrus can also be pharmacologically induced in female rats by subcutaneously injecting 10 &mu;g of &beta;-estradiol in 0.1 cc sterile sesame oil 48 hours prior to training followed by 500 &mu;g of progesterone in 0.1 cc sterile sesame oil 4 hours before training. Additionally, male rats that are naive to female rats must be sexually experienced prior to vocal training. This is accomplished by placing a female rat that is exhibiting signs of sexual receptivity (darting, lordosis, ear wiggling) into the male's home cage until the male mounts and ejaculates. Males may not mount during the first few sessions and the female may be removed after the male shows interest in the female by sniffing and/or chasing the female around the cage. Sexually experiencing is done once a day for two weeks. This time period also provides an opportunity for rats to habituate to the experimenter(s) and recording room.
One limitation to this method is the subjective determination of the vocalization type. In addition, this factor limits the speed of data collection, as each call must be independently examined and manually classified. Related to this is the importance of having experienced raters who have been consistently trained in vocalization identification. Both intra and inter-rater reliability should be assessed within your own lab.
In summary, ultrasonic vocalizations in rodents are used in many different models to study brain-behavior relationships, including emotional states, reward and addiction, and disease states, such as autism.6,14-16 This novel method is a way to study the mechanisms underlying targeted vocal training for the treatment of voice deficits in aging and Parkinson Disease. This method has the potential to be applied to other disease models which affect communication and voice.

<table border="1">
	<tbody>
 	<tr>
 	<th>Name</th>
 <th>Company</th>
 <th>Catalogue number</th>
 </tr>
 <tr>
 	<td>Condenser ultrasound microphone CM16/CMPA</td>
 <td>Avisoft Bioacoustics</td>
 <td>40011</td>
 </tr>
 <tr>
 	<td>UltraSoundGate 116Hb</td>
 <td>Avisoft Bioacoustics</td>
 <td>41161</td>
 </tr>
 <tr>
 	<td>Avisoft-RECORDER</td>
 <td>Avisoft Bioacoustics</td>
 <td>10201</td>
 </tr>

 <tr>
 	<td>Avisoft-SASLab Pro</td>
 <td>Avisoft Bioacoustics</td>
 <td>10101</td>
 </tr>
 </tbody>
</table>

targeted training of ultrasonic vocalizations in aged and parkinsonian rats

Voice deficits are a common complication of both Parkinson disease (PD) and aging; they can significantly diminish quality of life by impacting communication abilities. 1, 2 Targeted training (speech/voice therapy) can improve specific voice deficits,3, 4 although the underlying mechanisms of behavioral interventions are not well understood. Systematic investigation of voice deficits and therapy should consider many factors that are difficult to control in humans, such as age, home environment, age post-onset of disease, severity of disease, and medications. The method presented here uses an animal model of vocalization that allows for systematic study of how underlying sensorimotor mechanisms change with targeted voice training. The ultrasonic recording and analysis procedures outlined in this protocol are applicable to any investigation of rodent ultrasonic vocalizations.
The ultrasonic vocalizations of rodents are emerging as a valuable model to investigate the neural substrates of behavior.5-8 Both rodent and human vocalizations carry semiotic value and are produced by modifying an egressive airflow with a laryngeal constriction.9, 10 Thus, rodent vocalizations may be a useful model to study voice deficits in a sensorimotor context. Further, rat models allow us to study the neurobiological underpinnings of recovery from deficits with targeted training.
To model PD we use Long-Evans rats (Charles River Laboratories International, Inc.) and induce parkinsonism by a unilateral infusion of 7 &mu;g of 6-hydroxydopamine (6-OHDA) into the medial forebrain bundle which causes moderate to severe degeneration of presynaptic striatal neurons (for details see Ciucci, 2010).11, 12 For our aging model we use the Fischer 344/Brown Norway F1 (National Institute on Aging).
Our primary method for eliciting vocalizations is to expose sexually-experienced male rats to sexually receptive female rats. When the male becomes interested in the female, the female is removed and the male continues to vocalize. By rewarding complex vocalizations with food or water, both the number of complex vocalizations and the rate of vocalizations can be increased (Figure 1).
An ultrasonic microphone mounted above the male's home cage records the vocalizations. Recording begins after the female rat is removed to isolate the male calls. Vocalizations can be viewed in real time for training or recorded and analyzed offline. By recording and acoustically analyzing vocalizations before and after vocal training, the effects of disease and restoration of normal function with training can be assessed. This model also allows us to relate the observed behavioral (vocal) improvements to changes in the brain and neuromuscular system.

Watch this Scientific Journal Video about Targeted Training of Ultrasonic Vocalizations in Aged and Parkinsonian Rats at JoVE.com

Targeted Training of Ultrasonic Vocalizations in Aged and Parkinsonian Rats

Voice disorders are debilitating in aging and Parkinson disease. The ultrasonic vocalizations of rats, also affected by these conditions, can be used to study these voice disorders, their neural substrates, and the nature of functional recovery with behavioral intervention.

Voice deficits are a common complication of both Parkinson disease (PD) and aging; they can significantly diminish quality of life by impacting ...

targeted-training-ultrasonic-vocalizations-aged-parkinsonian

Research

JoVE Journal

Neuroscience

19.7K 조회수.  University of Wisconsin. 음성 장애는 노화와 파킨슨 질환에 쇠약하게하고 있습니다. 또한 이러한 조건에 의해 영향을 쥐의 초음파 vocalizations는, 이러한 음성 장애, 그들의 신경 기판, 그리고 행동의 개입과 기능 회복의 성격을 연구하는 데 사용할 수 있습니다.

비디오: Targeted Training of Ultrasonic Vocalizations in Aged and Parkinsonian Rats

Assessment of Ultrasonic Vocalizations During Drug Self-administration in Rats

Drug self-administration procedures are commonly used to study behavioral and neurochemical changes associated with human drug abuse, addiction and relapse. Various types of behavioral activity are commonly utilized as measures of drug motivation in animals. However, a crucial component of drug abuse relapse in abstinent cocaine users is "drug craving", which is difficult to model in animals, as it often occurs in the absence of overt behaviors. Yet, it is possible that a class of ultrasonic vocalizations (USVs) in rats may be a useful marker for affective responses to drug administration, drug anticipation and even drug craving. Rats vocalize in ultrasonic frequencies that serve as a communicatory function and express subjective emotional states. Several studies have shown that different call frequency ranges are associated with negative and positive emotional states. For instance, high frequency calls ("50-kHz") are associated with positive affect, whereas low frequency calls ("22-kHz") represent a negative emotional state. This article describes a procedure to assess rat USVs associated with daily cocaine self-administration. For this procedure, we utilized standard single-lever operant chambers housed within sound-attenuating boxes for cocaine self-administration sessions and utilized ultrasonic microphones, multi-channel recording hardware and specialized software programs to detect and analyze USVs. USVs measurements reflect emotionality of rats before, during and after drug availability and can be correlated with commonly assessed drug self-administration behavioral data such lever responses, inter-response intervals and locomotor activity. Since USVs can be assessed during intervals prior to drug availability (e.g., anticipatory USVs) and during drug extinction trials, changes in affect associated with drug anticipation and drug abstinence can also be determined. In addition, determining USV changes over the course of short- and long-term drug exposure can provide a more detailed interpretation of drug exposure effects on affective functioning.

Drug self-administration and ultrasonic vocalizations (USV) are used as behavioral assessments in animal research, but rarely in combination. The purpose of this article is to describe the advantages of recording USVs during drug self-administration procedures to assess affective responses to drug experience.

Drug self-administration procedures are commonly used to study behavioral and neurochemical changes associated with human drug abuse, addiction and ...

Kinematics and Ground Reaction Force Determination: A Demonstration Quantifying Locomotor Abilities of Young Adult, Middle-aged, and Geriatric Rats

Behavior, in its broadest definition, can be defined as the motor manifestation of physiologic processes. As such, all behaviors manifest through the motor system. In the fields of neuroscience and orthopedics, locomotion is a commonly evaluated behavior for a variety of disease models. For example, locomotor recovery after traumatic injury to the nervous system is one of the most commonly evaluated behaviors 1-3. Though locomotion can be evaluated using a variety of endpoint measurements (e.g. time taken to complete a locomotor task, etc), semiquantitative kinematic measures (e.g. ordinal rating scales (e.g. Basso Beattie and Bresnahan locomotor (BBB) rating scale, etc)) and surrogate measures of behaviour (e.g. muscle force, nerve conduction velocity, etc), only kinetics (force measurements) and kinematics (measurements of body segments in space) provide a detailed description of the strategy by which an animal is able to locomote 1. Though not new, kinematic and kinetic measurements of locomoting rodents is now more readily accessible due to the availability of commercially available equipment designed for this purpose. Importantly, however, experimenters need to be very familiar with theory of biomechanical analyses and understand the benefits and limitations of these forms of analyses prior to embarking on what will become a relatively labor-intensive study. The present paper aims to describe a method for collecting kinematic and ground reaction force data using commercially available equipment. Details of equipment and apparatus set-up, pre-training of animals, inclusion and exclusion criteria of acceptable runs, and methods for collecting the data are described. We illustrate the utility of this behavioral analysis technique by describing the kinematics and kinetics of strain-matched young adult, middle-aged, and geriatric rats.

Locomotion is often examined as a behavioural outcome in various models of disease in fields such as neuroscience and orthopedics. This video paper intends to describe a method for collecting ground reaction forces and kinematics from rats during unrestrained locomotion.

Behavior, in its broadest definition, can be defined as the motor manifestation of physiologic processes.  As such, all behaviors manifest through the ...

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 ...

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from individual neurons are critical for understanding how neural circuits function under natural conditions. Traditionally, these recordings have been performed 'blind', meaning the identity of the recorded cell is unknown at the start of the recording. Cellular identity can be subsequently determined via intracellular1, juxtacellular2 or loose-patch3 iontophoresis of dye, but these recordings cannot be pre-targeted to specific neurons in regions with functionally heterogeneous cell types. Fluorescent proteins can be expressed in a cell-type specific manner permitting visually-guided single-cell electrophysiology4-6. However, there are many model systems for which these genetic tools are not available. Even in genetically accessible model systems, the desired promoter may be unknown or genetically homogenous neurons may have varying projection patterns. Similarly, viral vectors have been used to label specific subgroups of projection neurons7, but use of this method is limited by toxicity and lack of trans-synaptic specificity. Thus, additional techniques that offer specific pre-visualization to record from identified single neurons in vivo are needed. Pre-visualization of the target neuron is particularly useful for challenging recording conditions, for which classical single-cell recordings are often prohibitively difficult8-11. The novel technique described in this paper uses retrograde transport of a fluorescent dye applied using tungsten needles to rapidly and selectively label a specific subset of cells within a particular brain region based on their unique axonal projections, thereby providing a visual cue to obtain targeted electrophysiological recordings from identified neurons in an intact circuit within a vertebrate CNS.
The most significant novel advancement of our method is the use of fluorescent labeling to target specific cell types in a non-genetically accessible model system. Weakly electric fish are an excellent model system for studying neural circuits in awake, behaving animals12. We utilized this technique to study sensory processing by "small cells" in the anterior exterolateral nucleus (ELa) of weakly electric mormyrid fish. "Small cells" are hypothesized to be time comparator neurons important for detecting submillisecond differences in the arrival times of presynaptic spikes13. However, anatomical features such as dense myelin, engulfing synapses, and small cell bodies have made it extremely difficult to record from these cells using traditional methods11, 14. Here we demonstrate that our novel method selectively labels these cells in 28% of preparations, allowing for reliable, robust recordings and characterization of responses to electrosensory stimulation.

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

Retrograde transport of fluorescent dye labels a sub-population of neurons based on anatomical projection. Labeled axons can be visually targeted in vivo, permitting extracellular recording from identified axons. This technique facilitates recording when neurons cannot be labeled through genetic manipulation or are difficult to isolate using 'blind' in vivo approaches.

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from ...

Functional Neuroimaging Using Ultrasonic Blood-brain Barrier Disruption and Manganese-enhanced MRI

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains technically challenging. One approach, Activation-Induced Manganese-enhanced MRI (AIM MRI), has been used successfully to map neuronal activity in rodents 1-5. In AIM MRI, Mn2+ acts a calcium analog and accumulates in depolarized neurons 6,7. Because Mn2+ shortens the T1 tissue property, regions of elevated neuronal activity will enhance in MRI. Furthermore, Mn2+ clears slowly from the activated regions; therefore, stimulation can be performed outside the magnet prior to imaging, enabling greater experimental flexibility. However, because Mn2+ does not readily cross the blood-brain barrier (BBB), the need to open the BBB has limited the use of AIM MRI, especially in mice.
One tool for opening the BBB is ultrasound. Though potentially damaging, if ultrasound is administered in combination with gas-filled microbubbles (i.e., ultrasound contrast agents), the acoustic pressure required for BBB opening is considerably lower. This combination of ultrasound and microbubbles can be used to reliably open the BBB without causing tissue damage 8-11.
Here, a method is presented for performing AIM MRI by using microbubbles and ultrasound to open the BBB. After an intravenous injection of perflutren microbubbles, an unfocused pulsed ultrasound beam is applied to the shaved mouse head for 3 minutes. For simplicity, we refer to this technique of BBB Opening with Microbubbles and UltraSound as BOMUS 12. Using BOMUS to open the BBB throughout both cerebral hemispheres, manganese is administered to the whole mouse brain. After experimental stimulation of the lightly sedated mice, AIM MRI is used to map the neuronal response.
To demonstrate this approach, herein BOMUS and AIM MRI are used to map unilateral mechanical stimulation of the vibrissae in lightly sedated mice 13. Because BOMUS can open the BBB throughout both hemispheres, the unstimulated side of the brain is used to control for nonspecific background stimulation. The resultant 3D activation map agrees well with published representations of the vibrissae regions of the barrel field cortex 14. The ultrasonic opening of the BBB is fast, noninvasive, and reversible; and thus this approach is suitable for high-throughput and/or longitudinal studies in awake mice.

A technique is described for broadly opening the blood-brain barrier in the mouse using microbubbles and ultrasound. Using this technique, manganese can be administered to the mouse brain. Because manganese is an MRI contrast agent that accumulates in depolarized neurons, this approach enables imaging of neuronal activity.

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains ...

Fluorescence and Bioluminescence Imaging of Subcellular Ca2+ in Aged Hippocampal Neurons

Susceptibility to neuron cell death associated to neurodegeneration and ischemia are exceedingly increased in the aged brain but mechanisms responsible are badly known. Excitotoxicity, a process believed to contribute to neuron damage induced by both insults, is mediated by activation of glutamate receptors that promotes Ca2+ influx and mitochondrial Ca2+ overload. A substantial change in intracellular Ca2+ homeostasis or remodeling of intracellular Ca2+ homeostasis may favor neuron damage in old neurons. For investigating Ca2+ remodeling in aging we have used live cell imaging in long-term cultures of rat hippocampal neurons that resemble in some aspects aged neurons in vivo. For this end, hippocampal cells are, in first place, freshly dispersed from new born rat hippocampi and plated on poli-D-lysine coated, glass coverslips. Then cultures are kept in controlled media for several days or several weeks for investigating young and old neurons, respectively. Second, cultured neurons are loaded with fura2 and subjected to measurements of cytosolic Ca2+ concentration using digital fluorescence ratio imaging. Third, cultured neurons are transfected with plasmids expressing a tandem of low-affinity aequorin and GFP targeted to mitochondria. After 24 hr, aequorin inside cells is reconstituted with coelenterazine and neurons are subjected to bioluminescence imaging for monitoring of mitochondrial Ca2+ concentration. This three-step procedure allows the monitoring of cytosolic and mitochondrial Ca2+ responses to relevant stimuli as for example the glutamate receptor agonist NMDA and compare whether these and other responses are influenced by aging. This procedure may yield new insights as to how aging influence cytosolic and mitochondrial Ca2+ responses to selected stimuli as well as the testing of selected drugs aimed at preventing neuron cell death in age-related diseases.

Fluorescence and Bioluminescence Imaging of Subcellular Ca2+ in Aged Hippocampal Neurons

Intracellular Ca2+ remodeling in aging may contribute to excitotoxicity and neuron damage, processes mediated by Ca2+ overload. We aimed at investigating Ca2+ remodeling in the aging brain using fluorescence and bioluminescence imaging of cytosolic and mitochondrial Ca2+ in long-term cultures of rat hippocampal neurons, a model of neuronal aging.

Susceptibility to neuron cell death associated to neurodegeneration and ischemia are exceedingly increased in the aged brain but mechanisms responsible ...

Sequential Extraction of Soluble and Insoluble Alpha-Synuclein from Parkinsonian Brains

Alpha-synuclein (&#945;-syn) protein is abundantly expressed mainly within neurons, and exists in a number of different forms - monomers, tetramers, oligomers and fibrils. During disease, &#945;-syn undergoes conformational changes to form oligomers and high molecular weight aggregates that tend to make the protein more insoluble. Abnormally aggregated &#945;-syn is a neuropathological feature of Parkinson&#39;s disease (PD), dementia with Lewy bodies (DLB) and multiple system atrophy (MSA). Biochemical characterization and analysis of insoluble &#945;-syn using buffers with increasing detergent strength and high-speed ultracentrifugation provides a powerful tool to determine the development of &#945;-syn pathology associated with disease progression. This protocol describes the isolation of increasingly insoluble/aggregated &#945;-syn from post-mortem human brain tissue. This methodology can be adapted with modifications to studies of normal and abnormal &#945;-syn biology in transgenic animal models harbouring different &#945;-syn mutations as well as in other neurodegenerative diseases that feature aberrant fibrillar deposits of proteins related to their respective pathologies.

Here, we present a protocol for the isolation of increasingly insoluble/aggregated alpha-synuclein (&#945;-syn) from post-mortem human brain tissue. Through the utilization of buffers with increasing detergent strength and high-speed ultracentrifugation techniques, the variable properties of &#945;-syn aggregation in diseased and non-diseased tissue can be examined.

Alpha-synuclein (&#945;-syn) protein is abundantly expressed mainly within neurons, and exists in a number of different forms - monomers, tetramers, ...

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 ...

Activity-based Training on a Treadmill with Spinal Cord Injured Wistar Rats

Spinal cord injury (SCI) results in lasting deficits that include both mobility and a multitude of autonomic-related dysfunctions. Locomotor training (LT) on a treadmill is widely used as a rehabilitation tool in the SCI population with many benefits and improvements to daily life. We utilize this method of activity-based task-specific training (ABT) in rodents after SCI to both elucidate the mechanisms behind such improvements and to enhance and improve upon existing clinical rehabilitation protocols. Our current goal is to determine the mechanisms underlying ABT-induced improvements in urinary, bowel, and sexual function in SCI rats after a moderate to severe level of contusion. After securing each individual animal in a custom-made adjustable vest, they are secured to a versatile body weight support mechanism, lowered to a modified three-lane treadmill and assisted in step-training for 58 minutes, once a day for 10 weeks. This setup allows for the training of both quadrupedal and forelimb-only animals, alongside two different non-trained groups. Quadrupedal-trained animals with body weight support are aided by a technician present to assist in stepping with proper hind limb placement as necessary, while forelimb-only trained animals are raised at the caudal end to ensure no hind limb contact with the treadmill and no weight-bearing. One non-trained SCI group of animals is placed in a harness and rests next to the treadmill, while the other control SCI group remains in its home cage in the training room nearby. This paradigm allows for the training of multiple SCI animals at once, thus making it more time-efficient in addition to ensuring that our pre-clinical animal model mimics the clinical representation as close as possible, particularly with respect to the body weight support with manual assistance.

This protocol demonstrates our model of activity-based locomotor treadmill training for rats with spinal cord injury (SCI). Included is both quadrupedal and forelimb-only groups, in addition to two distinct types of non-trained control groups. Investigators are able to assess training effects on SCI rats using this protocol.

Spinal cord injury (SCI) results in lasting deficits that include both mobility and a multitude of autonomic-related dysfunctions. Locomotor training (LT) ...

Measuring Statistical Learning Across Modalities and Domains in School-Aged Children Via an Online Platform and Neuroimaging Techniques

Statistical learning, a fundamental skill to extract regularities in the environment, is often considered a core supporting mechanism of the first language development. While many studies of statistical learning are conducted within a single domain or modality, recent evidence suggests that this skill may differ based on the context in which the stimuli are presented. In addition, few studies investigate learning as it unfolds in real-time, rather focusing on the outcome of learning. In this protocol, we describe an approach for identifying the cognitive and neural basis of statistical learning, within an individual, across domains (linguistic vs. non-linguistic) and sensory modalities (visual and auditory). The tasks are designed to cast as little cognitive demand as possible on participants, making it ideal for young school-aged children and special populations. The web-based nature of the behavioral tasks offers a unique opportunity for us to reach more representative populations nationwide, to estimate effect sizes with greater precision, and to contribute to open and reproducible research. The neural measures provided by the functional magnetic resonance imaging (fMRI) task can inform researchers about the neural mechanisms engaged during statistical learning, and how these may differ across individuals on the basis of domain or modality. Finally, both tasks allow for the measurement of real-time learning, as changes in reaction time to a target stimulus is tracked across the exposure period. The main limitation of using this protocol relates to the hour-long duration of the experiment. Children might need to complete all four statistical learning tasks in multiple sittings. Therefore, the web-based platform is designed with this limitation in mind so that tasks may be disseminated individually. This methodology will allow users to investigate how the process of statistical learning unfolds across and within domains and modalities in children from different developmental backgrounds.

Presented here is a protocol introducing a set of child-friendly statistical learning tasks geared towards examining children&#8217;s learning of temporal statistical patterns across domains and sensory modalities. The developed tasks collect behavioral data using the web-based platform and task-based functional magnetic resonance imaging (fMRI) data for examining neural engagement during statistical learning.