Name: assessment of ultrasonic vocalizations during drug self-administration in rats
Uploaded: 2010-07-22T17:00:00
Duration: 9 min 25 s
Description: Watch this Scientific Journal Video about Assessment of Ultrasonic Vocalizations During Drug Self-administration in Rats at JoVE.com

This work has been funded by NIH Grants RO1DA014640 and 3R01DA014640-05S1, The University of Texas VP Research Office (C.L.D), NIDA Drug Supply Program, and the University of Texas Waggoner Center for Alcohol and Addiction Research Bruce-Jones Graduate Fellowship (E.Y.M). We thank Leah McAleer, Mohamed Abdalla, Neha Thakore, Byron Barksdale, Tiffany Nguyen, Tian Tian, Hunter Owen, Proy Phongsawad, Helen Reed, Rachel Chavana, Linda Ju and Rosie Maddox for their assistance in USV data analyses.

*= important points <ol> <li>Well-handled male Sprague-Dawley rats are trained to lever press with food reward (45 mg sucrose pellets; Bio-Serv, Frenchtown, NJ).</li> <li>*Intravenous catheters are constructed out of stainless steel cannula (Plastics One, VA) and Silastic tubing. Rats undergo a jugular catheterization surgical procedure to allow drug self-administration. After surgery, catheter patency is maintained by daily flushing with 0.1 ml of a 0.9% saline, heparin and Timentin solution.</li> <li>Cocaine (NIDA Drug Inventory and Supply and Control Program; RTI International, Research Triangle Park, NC) used in this experiment was dissolved in isotonic saline solution (0.9%) in the appropriate dose concentrations according to animal weights (0.75 mg/kg/injection) so that each injection was 0.1 ml. Control animals received saline solution in the same volume/injection.</li> <li>*The apparatus used for drug self-administration consists of a single-lever operant chamber (28 x 22 x 21 cm) equipped with house and stimulus lights and 3 sets of photobeams. The operant chamber is housed within a larger sound-attenuating chamber (Med Associated, St. Albans, VT) to limit outside noise and light. During the administration sessions, the animals were intravenously connected via tubing to a syringe mounted on a motorized pump (Razel Scientific Instruments, Model A, St. Albans, VT) containing either a cocaine solution (0.75 mg/kg/injection) or sterile saline. After each lever press, a stimulus light above the lever illuminates and cocaine or saline is administered over a 6-sec infusion time. Locomotor activity is assessed as the number of photobeam interruptions. The drug self-administration program is controlled, and behavioral responses (e.g., lever responses and locomotor activity) are recorded using a Med Pentium computer equipped with Med PC software (Med Associates).</li> <li>*To detect and record USVs, ultrasonic microphones, data acquisition hardware, recording software (Avisoft Bioacoustics, Berlin, Germany) and PC computer systems are needed. For the current experiment, ultrasonic microphones (frequency response range =10-100-kHz) are securely mounted in the center of the operant chambers. Microphones are connected to data acquisition hardware (Avisoft-Ultrasoundgate) located outside of the operant chamber and recordings are collected on a PC computer system (Dell Optiplex GX270, Windows XP Professional, Intel(R) Pentium(R) 4 CPU, 2.60 GHz, 1 GB RAM) using RECORDER multi-channel recording software (Avisoft Bioacoustics).</li> <li>*Each drug self-administration/USV recording session proceeds as follows: <ol class="lalpha"> <li>The animals are gently taken out of their home cage and their indwelling intravenous catheter is flushed with 0.05 ml of a heparinized saline solution to detect possible catheter malfunctions.</li> <li>*Rats are connected to a swivel mounted on the operant chamber that allows either drug or vehicle administration during the self-administration sessions. Animals are gently placed into the operant chamber and the doors are quietly closed. The drug self-administration program (Med PC) and the USV recording software (RECORDER) are on two separate computer systems. Therefore, in order to be able to link behaviors within the operant chamber with USV detection, both programs need to be started simultaneously (e.g., may need two people depending on location of computers).</li> <li>*The individual USVs are recorded and time-stamped continuously throughout the session. The MedPC program controlling and recording events within the operant chamber can be written to any specification. The MedPC program automatically closes at the end of the session. The USV recording needs to be stopped manually on the USV recording-dedicated computer system.</li> <li>At the end of the session, the animals are gently taken out of the operant chamber, disconnected from the swivel and flushed with 0.05 ml heparinized saline to clear the catheter from drug residue.</li> </ol> </li> <li>*For logistical purposes, we used a separate computer system for USV analyses (Dell Optiplex 745, Windows XP Professional, Intel(R) Core(TM), 2 CPU, 2.40 GHz, 2 GB RAM), but the analyzing software can also be installed on the same computer system as the recording software. USV analyses must be performed carefully to ensure accuracy: <ol class="lalpha"> <li>*SASLab Pro analyzing software (Avisoft Bioacoustics, Berlin, Germany) allows the recorded USVs to be visible and audible for the human experimenters displayed in a spectogram.</li> <li>*Using the available configuration settings, the resulting spectogram can be modified for individual preferences. In addition, background noises in low frequencies (e.g., generated by the fan of the operant chambers) can be cut out to allow the software to automatically detect sounds above that frequency in the spectogram. These functions assist in data analyses as by making the sound files shorter and faster to assess.</li> <li>Background noises in the same frequencies as USVs are sometimes detected in the spectrogram. Since the sound files are recorded as .wav files, the researcher needs to listen to the sound files during assessment to confirm and differentiate USVs from background noise.</li> <li>*The analyzing software can create Excel files with parameters such as call frequencies, bandwidth, duration, and exact time of the emitted USV. The latter allows the correlation of USVs to the exact time before or after drug is self-administered. This allows USVs to be used as real-time measurements of the rat's emotional response at specific instances during the experimental procedure.</li> </ol> </li> </ol> Representative Results: <img src="/files/ftp_upload/2041/2041fig1.jpg" alt="Figure 1" /> Figure 1: Spectogram display of a 22-kHz range USV. Long calls in the 22-kHz range are elicited in conjunction with aversive stimuli and are thought to reflect a negative emotional state. <img src="/files/ftp_upload/2041/2041fig2.jpg" alt="Figure 2" /> Figure 2: Spectogram display of 50-kHz USVs. Short "flat" and "frequency modulated" 50-kHz USVs are elicited in anticipation and during cocaine self-administration. They are associated with positive affect. <img src="/files/ftp_upload/2041/2041fig3.jpg" alt="Figure 3" /> Figure 3: Cocaine-Induced 50-kHz range USVs. Graph depicts USVs (10-sec bins) elicited before and after self-administered cocaine injections (0.75 mg/kg/inj) in a representative rat on Day 5 of cocaine self-administration sessions. Immediately after placement within the operant chamber and prior to cocaine availability, cocaine-anticipatory USVs were elicited and during self-administration, cocaine-induced USVs were emitted.

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	<li>Brudzynski, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Communication+of+adult+rats+by+ultrasonic+vocalization:+biological,+sociobiological,+and+neuroscience+approaches.">Communication of adult rats by ultrasonic vocalization: biological, sociobiological, and neuroscience approaches.</a> ILAR J. 50 (1), 43-43 (2009).</li><li>Knutson, B., Burgdorf, J., Panksepp, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrasonic+vocalizations+as+indices+of+affective+states+in+rats.">Ultrasonic vocalizations as indices of affective states in rats.</a> Psychol Bull. 128 (6), 961-961 (2002).</li><li>Wohr, M., Schwarting, R. 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No conflicts of interest declared.

Rats vocalize in ultrasonic frequencies that serve a social and communicatory function and express subjective emotional states 1,2. 22-kHz USVs are triggered by events that cause anxiety and distress, such as footshock cues, the presence of predators, aversive drugs and social defeat 2,3. Therefore, these typically long USVs (&gt;0.3s) are thought to represent a state of negative affect of the animal. Conversely, shorter 50-kHz USVs (&gt;0.3s) are emitted during and in anticipation of positive encounters, such as feeding, mating and social play, and have thus been linked to a positive emotional state of the animal 2,4. These calls have also been associated with reinforcing events related to the activation of the dopaminergic system 5. For example, increased 50-kHz USVs have been elicited by systemic injections of cocaine 6 or d-amphetamine 7 and environments previously paired with drugs of abuse 8-10.
It has been proposed that increases in locomotor activity in rodents relate to enhanced motivation and appetitive behavior 11-14. However, this relationship is indirect as best. By studying the emergence and persistence of USVs of particular frequencies, it is possible to gain insight to emotional effects of various aspects of drug experience.
In this experimental setup, we collected USV recordings immediately before and during drug self-administration sessions. This allowed us to examine the development of drug expectation during the non-drug interval just prior to the start of the session and to assess the direct effects of cocaine on USVs. The drug self-administration method allows rats to directly control their own drug intake levels animals and develop accurate expectations of the timing and frequency of drug administration. As a result, USVs detected during these sessions reflect accurate expectations of drug delivery. In addition, USVs can be assessed during intervals prior to drug availability (e.g., anticipatory USVs), in rats receiving experimenter-administered drugs, and during drug extinction trials. Therefore, changes in affect associated with drug anticipation, non-contingent drug administration and drug abstinence can also be determined. Analyses of USV changes over the course of short- and long-term drug exposure could provide a more detailed interpretation of drug exposure effects on affective functioning.
In addition to drug self-administration experiments, USV recordings in operant chambers can also be used for a variety of other behavioral manipulations associated with reward or aversion, such as alcohol drinking, food reinforcement, and fear conditioning and extinction procedures.
Important Methodological Details
Since the MedPC software can be used to precisely manipulate experimental conditions, such as lever availability and lighting conditions, its use in combination with USV recording and analyses allows emotional effects of drug-associated cue presentations (i.e., light presentations or other sensory stimuli) and the direct effects of drug intake to be determined. Therefore, to ensure overlapping timelines of self-administration procedures and USV recordings, it is crucial that both software programs (e.g., MedPC and RECORDER) are activated simultaneously at the start of each session.
Due to the enormous amount of digital space required to store USV data (e.g., a 10-minute sound file requires approx 250 MB), caution must be taken when multiple animals are run on a single computer system at the same time. To avoid recording errors, the RAM size of the recording computer system needs be substantial (1-2 GB). Other ways to decrease recording errors include lowering the USV resolution during recording and/or reduce the number of animals run at one time. In addition, it is important that the researcher is vigilant to all aspects of data collection throughout the experimental procedure. For instance, the accumulation of a large amount of recording data on the hard drive may cause the USV recording program to pause. In this case, the recording program would need to be restarted immediately to avoid loss of USV data and de-synchronization of USVs with other behavioral measures.
Environmental conditions may influence USV calling. For example, the touch by an unfamiliar human can cause the animal to emit negative 22-kHz calls, which could be mistaken as an aversion to the experimental conditions. Therefore, intensive handling prior to experimental manipulations is an important aspect of behavioral experiments. In addition, since novel environments can also elicit non-specific USV calls, animals should be habituated to the test environment if possible.

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		<th>Comment</th>
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<tr>
<td>Operant Conditioning Behavior (Drug Self-administration) Test Package for Rat</td>
<td>&nbsp;</td>
<td><a href="http://www.med-associates.com/">Med Associates, Inc</a></td>
<td>MED-008-CT-B1</td>
<td>&nbsp;</td>
<tr>
<td>Infrared Source and Detector (Photobeams)</td>
<td>&nbsp;</td>
<td><a href="http://www.med-associates.com/">Med Associates, Inc</a></td>
<td>ENV-253SD ENV-253</td>
<td>&nbsp;</td>
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<td>Med PC Software</td>
<td>&nbsp;</td>
<td><a href="http://www.med-associates.com/">Med Associates, Inc</a></td>
<td>SOF-735</td>
<td>&nbsp;</td>
<tr>
<td>Single speed syringe pump</td>
<td>&nbsp;</td>
<td>Razel Scientific Instruments</td>
<td>Model R-E</td>
<td>&nbsp;</td>
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<td>45 mg sucrose pellets</td>
<td>&nbsp;</td>
<td>Bio-Serv</td>
<td>F0042</td>
<td>&nbsp;</td>
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<td>Catheter cannula</td>
<td>&nbsp;</td>
<td>Plastics One</td>
<td>C313G-5UP</td>
<td>&nbsp;</td>
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<td>Avisoft-UltraSoundGate 416H</td>
<td>&nbsp;</td>
<td>Avisoft BioAcoustics</td>
<td>Part #34163</td>
<td>&nbsp;</td>
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<td>Ultrasonic microphones</td>
<td>&nbsp;</td>
<td>Avisoft BioAcoustics</td>
<td>Part #40011</td>
<td>&nbsp;</td>
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<td>Avisoft-RECORDER Software</td>
<td>&nbsp;</td>
<td>Avisoft BioAcoutics</td>
<td>Part #10302</td>
<td>&nbsp;</td>
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<td>Avisoft-SASLab Software</td>
<td>&nbsp;</td>
<td>Avisoft BioAcoutics</td>
<td>Part #10101</td>
<td>&nbsp;</td>
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<td>PC Computer for USV recording</td>
<td>&nbsp;</td>
<td>Dell Optiplex GX270, Windows XP Professional, Intel (R) Pentium (R), 4 CPU, 2.60 GHz, 1 GB RAM</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>PC Computer for USV data analyses</td>
<td>&nbsp;</td>
<td>Dell Optiplex 745, Windows XP Professional Intel(R) Core (TM), 2 CPU, 2.40 GHz, 2GB RAM</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Cocaine</td>
<td>&nbsp;</td>
<td>RTI International</td>
<td>&nbsp;</td>
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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.

Watch this Scientific Journal Video about Assessment of Ultrasonic Vocalizations During Drug Self-administration in Rats at JoVE.com

Assessment of Ultrasonic Vocalizations During Drug Self-administration in Rats

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 ...

assessment-ultrasonic-vocalizations-during-drug-self-administration

Research

JoVE Journal

Neuroscience

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.

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 ...

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 ...

Methods for Intravenous Self Administration in a Mouse Model

Animal models have been developed to study the reinforcing effects of drugs, including the intravenous self-administration (IVSA) paradigm. The advantages of using an IVSA paradigm to study the reinforcing properties of drugs of abuse such as cocaine include the fact that the drug is self-administered instead of experimenter-administered, the schedule of reinforcement can be altered, and accurate measurement of the quantities of drug consumed as well as the timing and pattern of IV injections can be obtained. Furthermore, the intravenous route of administration avoids potential confounds related to first pass metabolism or taste, and produces rapid increases in blood and brain drug levels. As outlined in this video, intravenous self-administration can be obtained without prior food restriction or prior drug training following careful catheter placement during surgery and meticulous daily catheter flushing and maintenance. Experimental procedures outlined in this paper include a description of animal housing and acclimation methods, operant training using sweetened milk solutions, and catheter implantation surgery.

The intravenous self-administration (IVSA) paradigm is considered to be the gold standard in examining the reinforcing properties of drugs of abuse in rodents. This manuscript outlines the experimental procedures and surgical techniques necessary to obtain reliable IVSA data. In particular, meticulous catheter implantation and maintenance are highlighted.

Animal models have been developed to study the reinforcing effects of drugs, including the intravenous self-administration (IVSA) paradigm. The advantages ...

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 ...

Microdialysis of Ethanol During Operant Ethanol Self-administration and Ethanol Determination by Gas Chromatography

Operant self-administration methods are commonly used to study the behavioral and pharmacological effects of many drugs of abuse, including ethanol. However, ethanol is typically self-administered orally, rather than intravenously like many other drugs of abuse. The pharmacokinetics of orally administered drugs are more complex than intravenously administered drugs. Because understanding the relationship between the pharmacological and behavioral effects of ethanol requires knowledge of the time course of ethanol reaching the brain during and after drinking, we use in vivo microdialysis and gas chromatography with flame ionization detection to monitor brain dialysate ethanol concentrations over time. 
Combined microdialysis-behavioral experiments involve the use of several techniques. In this article, stereotaxic surgery, behavioral training and microdialysis, which can be adapted to test a multitude of self-administration and neurochemical centered hypotheses, are included only to illustrate how they relate to the subsequent phases of sample collection and dialysate ethanol analysis. Dialysate ethanol concentration analysis via gas chromatography with flame-ionization detection, which is specific to ethanol studies, is described in detail. Data produced by these methods reveal the pattern of ethanol reaching the brain during the self-administration procedure, and when paired with neurochemical analysis of the same dialysate samples, allows conclusions to be made regarding the pharmacological and behavioral effects of ethanol.

A method to determine the time course of ethanol concentration in the brains of rats during operant ethanol self-administration is described. Gas chromatography with flame ionization detection is used to quantify ethanol in the dialysate samples, because it has the sensitivity required for the small volumes that are generated.

Operant self-administration methods are commonly used to study the behavioral and pharmacological effects of many drugs of abuse, including ethanol.  ...

Unilateral Pyramidotomy of the Corticospinal Tract in Rats for Assessment of Neuroplasticity-inducing Therapies

The corticospinal tract (CST) can be completely severed unilaterally in the medullary pyramids of the rodent brainstem. The CST is a motor tract that has great importance for distal muscle control in humans and, to a lesser extent, in rodents. A unilateral cut of one pyramid results in loss of CST innervation of the spinal cord mainly on the contralateral side of the spinal cord leading to transient motor disability in the forelimbs and sustained loss of dexterity. Ipsilateral projections of the corticospinal tract are minor. We have refined our surgical method to increase the chances of lesion completeness. We describe postsurgical care. Deficits on the Montoya staircase pellet reaching test and the horizontal ladder test shown here are detected up to 8 weeks postinjury. Deficits on the cylinder rearing test are only detected transiently. Therefore, the cylinder test may only be suitable for detection of short term recovery. We show how, electrophysiologically and anatomically, one may assess lesions and plastic changes. We also describe how to analyse fibers from the uninjured CST sprouting across the midline into the deprived areas. It is challenging to obtain &#62;90% complete lesions consistently due to the proximity to the basilar artery in the medulla oblongata and survival rates can be low. Alternative surgical approaches and behavioural testing are described in this protocol. The pyramidotomy model is a good tool for assessing neuroplasticity-inducing treatments, which increase sprouting of intact fibers after injury.

The corticospinal tract, one of the major sensorimotor tracts, can be lesioned unilaterally in the rodent brainstem in order to test neuroplasticity-inducing therapies for the central nervous system. This surgical procedure (&#8220;pyramidotomy&#8221;) and postoperative assessments are described in this protocol.

The corticospinal tract (CST) can be completely severed unilaterally in the medullary pyramids of the rodent brainstem. The CST is a motor tract that has ...

Assessment of Neuronal Viability Using Fluorescein Diacetate-Propidium Iodide Double Staining in Cerebellar Granule Neuron Culture

Primary cultured Cerebellar Granule Neurons (CGNs) have been widely used as an in vitro model in neuroscience and neuropharmacology research. However, the co-existence of glial cells and neurons in CGN culture might lead to biases in the accurate assessment of neuronal viability. Fluorescein diacetate (FDA) and Propidium Iodide (PI) double staining has been used to measure cell viability by simultaneously evaluating the viable and dead cells. We used FDA-PI double staining to improve the sensitivities of the colorimetric assays and to evaluate neuronal viability in CGNs. Furthermore, we added blue fluorescent DNA stains (e.g., Hoechst) to improve the accuracy. This protocol describes how to improve the accuracy of assessment of neuronal viability by using these methods in CGN culture. Using this protocol, the number of glial cells can be excluded by using fluorescence microscopy. A similar strategy can be applied to distinguish the unwanted glial cells from neurons in various mixed cell cultures, such as primary cortical culture and hippocampal culture.

This protocol describes how to accurately measure neuronal viability using Fluorescein diacetate (FDA) and Propidium Iodide (PI) double staining in cultured cerebellar granule neurons, a primary neuronal culture used as an in vitro model in neuroscience and neuropharmacology research.

Primary cultured Cerebellar Granule Neurons (CGNs) have been widely used as an in vitro model in neuroscience and neuropharmacology research. However, the ...

Quantitative Cell Biology of Neurodegeneration in Drosophila Through Unbiased Analysis of Fluorescently Tagged Proteins Using ImageJ

With the rising prevalence of neurodegenerative diseases, it is increasingly important to understand the underlying pathophysiology that leads to neuronal dysfunction and loss. Fluorescence-based imaging tools and technologies enable unprecedented analysis of subcellular neurobiological processes, yet there is still a need for unbiased, reproducible, and accessible approaches for extracting quantifiable data from imaging studies. We have developed a simple and adaptable workflow to extract quantitative data from fluorescence-based imaging studies using Drosophila models of neurodegeneration. Specifically, we describe an easy-to-follow, semi-automated approach using Fiji/ImageJ to analyze two cellular processes: first, we quantify protein aggregate content and profile in the Drosophila optic lobe using fluorescent-tagged mutant huntingtin proteins; and second, we assess autophagy-lysosome flux in the Drosophila visual system with ratiometric-based quantification of a tandem fluorescent reporter of autophagy. Importantly, the protocol outlined here includes a semi-automated segmentation step to ensure all fluorescent structures are analyzed to minimize selection bias and to increase resolution of subtle comparisons. This approach can be extended for the analysis of other cell biological structures and processes implicated in neurodegeneration, such as proteinaceous puncta (stress granules and synaptic complexes), as well as membrane-bound compartments (mitochondria and membrane trafficking vesicles). This method provides a standardized, yet adaptable reference point for image analysis and quantification, and could facilitate reliability and reproducibility across the field, and ultimately enhance mechanistic understanding of neurodegeneration.

Quantitative Cell Biology of Neurodegeneration in Drosophila Through Unbiased Analysis of Fluorescently Tagged Proteins Using ImageJ

We have developed a simple and adaptable workflow to extract quantitative data from fluorescence-imaging-based cell biological studies of protein aggregation and autophagic flux in the central nervous system of Drosophila models of neurodegeneration.

With the rising prevalence of neurodegenerative diseases, it is increasingly important to understand the underlying pathophysiology that leads to neuronal ...

Microdialysis of Excitatory Amino Acids During EEG Recordings in Freely Moving Rats

Microdialysis is a well-established neuroscience technique that correlates the changes of neurologically active substances diffusing into the brain interstitial space with the behavior and/or with the specific outcome of a pathology (e.g., seizures for epilepsy). When studying epilepsy, the microdialysis technique is often combined with short-term or even long-term video-electroencephalography (EEG) monitoring to assess spontaneous seizure frequency, severity, progression and clustering. The combined microdialysis-EEG is based on the use of several methods and instruments. Here, we performed in vivo microdialysis and continuous video-EEG recording to monitor glutamate and aspartate outflow over time, in different phases of the natural history of epilepsy in a rat model. This combined approach allows the pairing of changes in the neurotransmitter release with specific stages of the disease development and progression. The amino acid concentration in the dialysate was determined by liquid chromatography. Here, we describe the methods and outline the principal precautionary measures one should take during in vivo microdialysis-EEG, with particular attention to the stereotaxic surgery, basal and high potassium stimulation during microdialysis, depth electrode EEG recording and high-performance liquid chromatography analysis of aspartate and glutamate in the dialysate. This approach may be adapted to test a variety of drug or disease induced changes of the physiological concentrations of aspartate and glutamate in the brain. Depending on the availability of an appropriate analytical assay, it may be further used to test different soluble molecules when employing EEG recording at the same time.

Here, we describe a method for in vivo microdialysis to analyze aspartate and glutamate release in the ventral hippocampus of epileptic and non-epileptic rats, in combination with EEG recordings. Extracellular concentrations of aspartate and glutamate may be correlated with the different phases of the disease.

Microdialysis is a well-established neuroscience technique that correlates the changes of neurologically active substances diffusing into the brain ...

Multifactorial Assessment of Motor Behavior in Rats after Unilateral Sciatic Nerve Crush Injury

The induction of a peripheral nerve injury is a widely used method in neuroscience for the assessment of repair and pain mechanisms among others. In addition, in the research field of movement disorders, sciatic crush injury has been employed to trigger a dystonia-like phenotype in genetically predisposed DYT-TOR1A rodent models of dystonia. To achieve consistent, reproducible and comparable results after a sciatic nerve crush injury, a standardized method for inducing the nerve crush is essential, in addition to a standardized phenotypical characterization. Attention must be paid not only to the specific assortment of behavioral tests, but also to the technical requirements, the correct execution and consecutive data analysis. This protocol describes in detail how to perform a sciatic nerve crush injury and provides a behavioral test battery for the assessment of motor deficits in rats that includes the open field test, the CatWalk XT gait analysis, the beam walking task, and the ladder rung walking task.

We provide a protocol for the assessment of motor behavior via a behavioral test battery in rats after sciatic nerve crush injury.

The induction of a peripheral nerve injury is a widely used method in neuroscience for the assessment of repair and pain mechanisms among others. In ...