This work was supported by the Natural Science Foundation of China (31171355) and the Natural Science Foundation of Guangdong (S2011010003403, 2014A030313440).

Animal care, surgical procedure, and recording procedures were in accordance with the guidelines for the South China Normal University Animal Care and Use Committee.
1. Electrode Preparation (Figure 1A-C)
NOTE: Computer pin loci is simply a pronged contact as a part of signal interface in communication devices. It consists of a male connector that plugs into the female connector.
<ol>
	<li>Carefully separate the male and female pins from the computer pin loci (Figure 1A) with the help of a pincer. Connect the male and female pins together to form an electrode and apply cyanoacrylate&#160;to create a strong adhesive bond (Figure 1C).</li>
	<li>Put the electrodes into a beaker filled with distilled water for and place it to ultrasonic cleaner for&#160;10 min. Move them to a drying oven at 45 &#176;C for 30 min. Sterilize the electrodes using UV light for 30 min.</li>
</ol>
2. Surgical procedure (Figure 1D-F)
<ol>
	<li>Prepare the sterilized surgical instruments and stereotaxic apparatus. Anesthetize the neonatal rat pup using isoflurane anesthesia (2.5 %) with air. When the pup is deeply anesthetized, adjust the dose of isoflurane to 1.0%. Perform a tail or toe pinch prior to the surgery to ensure the proper depth of anesthesia.</li>
	<li>Fix the head of the pup in the stereotaxic apparatus by placing the ear bars into the ear canals and slightly tightening them. 
	NOTE: Do not excessively tighten the ear bars, as the neonatal skull is very soft.</li>
	<li>Maintain the sterile surgical field by spraying all equipment with 70 % ethanol. Make a 15-mm incision on the head using a scalpel. Using forceps, gently pull the scalp away from the midline at the four corners. Put some saline-soaked cotton below the skin to keep the incision wide open (Figure 1D).</li>
	<li>Find the bregma and lambda points on the skull and mark them with a pencil. Use a syringe needle (26 G) to make two burr holes into the prefrontal cortex (PFC) and hippocampus. 
	NOTE: The PFC is located at +1.8 mm posterior to bregma and -0.5 mm lateral to the midline, while the hippocampus is located at -2.0 mm anterior to bregma and &#177;0.5 mm lateral to the midline (Figure 1D and E). The depth of the electrode should not be more than 2 mm below the cortical surface to minimize brain damage.&#160;</li>
	<li>Use forceps to hold the electrodes and insert the reference and recording electrodes into the PFC and hippocampus, respectively. Apply erythromycin ointment around the electrode to avoid any possible infection. Fix the electrode using cyanoacrylate.</li>
	<li>Prepare dental acrylic cement so that it has a gluey and viscous consistency. Apply the dental cement to cover the electrodes and the rest of the skull. 
	NOTE: Thoroughly dry the skull before applying the dental cement.</li>
	<li>Apply 5% picric acid onto the electrodes to protect them. 
	NOTE: The entire procedure should be done in a biosafety hood to maintain sterile conditions.</li>
	<li>Remove the animal from the stereotaxic frame and inject 300 &#181;L of 10 % glucose subcutaneously. Place it on a heated blanket for recovery. Make sure that the animal is warm (37 &#176;C) and ambulatory (i.e., completely recovered). Administer buprenorphine intraperitoneally (0.05 mg/kg) for post-surgical pain. 
	NOTE: Do not leave an animal unattended until it has regained sufficient consciousness (i.e. normal behavior and movement).</li>
	<li>Return the pup to its home cage with their dam after it regains consciousness. Wait for two days until the animal is fully recovered.</li>
</ol>
3. EEG Recording
<ol>
	<li>After full recovery, connect the electrodes implanted on the skull of the pup to the amplifier in its own cage. Connect the amplifier to an analogue-to-digital converter and attach the converter to a computer; the connecting lines should be treated carefully so that they do not get tangled.</li>
	<li>Select at least 10,000 Hz sampling rate on the data acquisition unit for recording (the bandwidth of the transmitter is 1-100 Hz). Ensure that the data is sampled properly.</li>
	<li>After getting the baseline recording, inject the pup intraperitoneally with kainic acid (KA) (2 mg/kg) to induce epileptic seizures. 15 min after the KA injection observe and record the epileptic discharges.&#160;Seizure produce through KA are usually physical. 
	NOTE:&#160;The ictal-tonic duration is about 15.2 &#177;0.9 s, seizure duration is about 62&#177;5 s. Seizures can be prevented in the&#160;neonatal rat by giving IP injection of chloral hydrate (400 mg/kg).</li>
	<li>Save the digitized data and analyze it using signal-processing software packages, such as spike2. Reveal the power level of different frequency components in the neonatal EEG signal by performing a power spectrum analysis. Calculate the power in a 1-min timeframe by finding the root-mean-square amplitude from 1 to 100 Hz (EEG band) 9.</li>
</ol>

<ol>
	<li>Pereda, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrical+synapses+and+their+functional+interactions+with+chemical+synapses.">Electrical synapses and their functional interactions with chemical synapses.</a> Nat Rev Neurosci. 15 (4), 250-263 (2014).</li><li>Chorev, E., Epsztein, J., Houweling, A. R., Lee, A. K., Brecht, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophysiological+recordings+from+behaving+animals--going+beyond+spikes.">Electrophysiological recordings from behaving animals--going beyond spikes.</a> Curr Opin Neurobiol. 19 (5), 513-519 (2009).</li><li>Freeborn, D. L., McDaniel, K. L., Moser, V. C., Herr, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+electroencephalography+(EEG)+to+assess+CNS+changes+produced+by+pesticides+with+different+modes+of+action:+effects+of+permethrin,+deltamethrin,+fipronil,+imidacloprid,+carbaryl,+and+triadimefon.">Use of electroencephalography (EEG) to assess CNS changes produced by pesticides with different modes of action: effects of permethrin, deltamethrin, fipronil, imidacloprid, carbaryl, and triadimefon.</a> Toxicol Appl Pharmacol. 282 (2), 184-194 (2015).</li><li>Werhahn, K. J., Hartl, E., Hamann, K., Breimhorst, M., Noachtar, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Latency+of+interictal+epileptiform+discharges+in+long-term+EEG+recordings+in+epilepsy+patients.">Latency of interictal epileptiform discharges in long-term EEG recordings in epilepsy patients.</a> Seizure. 29, 20-25 (2015).</li><li>Staba, R. J., Stead, M., Worrell, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophysiological+biomarkers+of+epilepsy.">Electrophysiological biomarkers of epilepsy.</a> Neurotherapeutics. 11 (2), 334-346 (2014).</li><li>Nimmrich, V., Draguhn, A., Axmacher, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+Network+Oscillations+in+Neurodegenerative+Diseases.">Neuronal Network Oscillations in Neurodegenerative Diseases.</a> Neuromolecular Med. 17 (3), 270-284 (2015).</li><li>Zayachkivsky, A., Lehmkuhle, M. J., Dudek, F. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+Continuous+EEG+Monitoring+in+Small+Rodent+Models+of+Human+Disease+Using+the+Epoch+Wireless+Transmitter+System.">Long-term Continuous EEG Monitoring in Small Rodent Models of Human Disease Using the Epoch Wireless Transmitter System.</a> J Vis Exp. (101), e52554 (2015).</li><li>Zayachkivsky, A., Lehmkuhle, M. J., Fisher, J. H., Ekstrand, J. J., Dudek, F. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recording+EEG+in+immature+rats+with+a+novel+miniature+telemetry+system.">Recording EEG in immature rats with a novel miniature telemetry system.</a> J Neurophysiol. 109 (3), 900-911 (2013).</li><li>Dzhala, V. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NKCC1+transporter+facilitates+seizures+in+the+developing+brain.">NKCC1 transporter facilitates seizures in the developing brain.</a> Nat Med. 11 (11), 1205-1213 (2005).</li><li>Tucker, A. M., Aquilina, K., Chakkarapani, E., Hobbs, C. E., Thoresen, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+amplitude-integrated+electroencephalography+and+interburst+interval+in+the+rat.">Development of amplitude-integrated electroencephalography and interburst interval in the rat.</a> Pediatr Res. 65 (1), 62-66 (2009).</li><li>Savard, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Involvement+of+neuronal+IL-1beta+in+acquired+brain+lesions+in+a+rat+model+of+neonatal+encephalopathy.">Involvement of neuronal IL-1beta in acquired brain lesions in a rat model of neonatal encephalopathy.</a> J Neuroinflammation. 10, 110 (2013).</li><li>Cuaycong, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+approach+to+the+study+of+hypoxia-ischemia-induced+clinical+and+subclinical+seizures+in+the+neonatal+rat.">A novel approach to the study of hypoxia-ischemia-induced clinical and subclinical seizures in the neonatal rat.</a> Dev Neurosci. 33 (3-4), 241-250 (2011).</li></ol>

The authors declare no conflict of interest.

Here, we report surgical and recording procedures to acquire EEG in freely moving neonatal rat pups by wired method (Figure 3). It has been suggested that the P7-P12 rat pup is at the developmental age that corresponds to a full-term human neonate 10,11. It is technically difficult to obtain high-quality EEG recording data when working with rat pups in this age group. In addition, it requires specialized handling and great care 8.
Previous studies examining in vivo EEG recordings in neonatal rats have been conducted using wired recording solutions in P12 or older animals 12, but these approaches are very costly. By relying on simple electrodes, we have been able to make an electrode (Figure 1) that reduces the cost of the experiment to as little as $1 while still giving high-density EEG recordings. Surgical implantation in this method can take as little as 10 min, depending on the complexity of the surgery.
The critical steps in this protocol is the implantation of electrodes. It should be done carefully. To maintain constant contact of the electrode with the skull, the following steps are important to consider. Firstly, remove the tissue debris on the skull with the help of sterilized cotton swab because it may set apart the electrode from the skull. Secondly, use cyanoacrylate&#160;properly to attach the electrode with the skull. Thirdly, keep the adequate viscosity of dental cement. It is better to apply dental cement in two layers; first layer should be a little dense to cover the skull while the second layer should be a little runny to cover the corners of first layer. Tap the dental cement gently with a pipette tip to settle it down to make the strong adhesive bond. If it is too watery it may build an insulating layer under the electrode, and if it is too dense, the dental cement will easily fall off from the skull due to its own weight. It is also important to maintain the pup&#39;s body temperature at 37 &#176;C throughout the surgery for survival.
The application of this procedure is limited to recording EEG in neonatal rat pups between the ages of P8 and P15 because the growing pups may remove the dental cement and electrode from their skull. In terms of further applications, this method can also be used to record the electrical activity of adult rats and mice after minor modification. Moreover, this method lends itself well to use in various behavioral experiments, such as the elevated plus maze.

It is well established that continuous communication between neurons is required to obtain normal brain function. The interneuronal communication primarily takes place at synapses, where information from one neuron is conveyed to a second neuron. This synaptic transmission is mediated by two types of dedicated structural arrangements: electrical or chemical synapses 1. Electrophysiology is the field that captures the electrical potential produced during interneuronal communication that control overall body functions and behavior 2. EEG is the most commonly used method among many electrophysiological techniques.
EEG is a technique used to detect changes in electrical signals produced by internal or external stimuli. Moreover, it is an essential test for clinical diagnosis and outcome prediction of various neurological conditions such as epilepsy, Parkinson&#39;s and Alzheimer&#39;s disease, as well as effects of pharmacological and toxicological agents 3. Generally, an epileptic patient shows hyperexcitability and impaired functional connectivity within the brain; these are summarized as interictal epileptiform discharges (IEDs) and can be recorded by EEG in the form of sharp, transient spikes; sharp waves; spike-wave complexes; or polyspikes 4. The main feature of the epileptic brain is the spontaneous occurrence of epileptic seizures, which can be recorded either from the scalp or from the brain parenchyma in order to locate the brain area responsible for the seizures 5. Furthermore, EEG also has very important implications in neurodegenerative disorders like Alzheimer&#39;s disease (AD). Research suggests that altered EEG recordings and impaired oscillatory networks in AD patients are common. However, our knowledge about the pathophysiology of network oscillations in neurodegenerative diseases is surprisingly incomplete and needs to be further explored 6.
In this protocol, we have designed a simple electrode with which one can record EEG to understand the electrical communication in both the normal and the pathological brain. The surgical implantation in this method is cheaper than other available procedures 7. Moreover, this method can be used to record high-quality and stable EEG signals for longer timeframes (i.e., 2-4 h every day for 1 week). In addition, we used lighter electrodes (weighing approximately 26 mg) that enable the animals to behave more naturally 8. This method is widely applicable to the study of EEG in neonatal rat pups that requires the amplifier and digitizer, commonly used in electrophysiology lab and does not require any additional devices.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Computer pin</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pincer</td>
			<td>DELI Group Co., Ltd.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>502 super glue</td>
			<td>DELI Group Co., Ltd.</td>
			<td>7144</td>
			<td></td>
		</tr>
		<tr>
			<td>Drying oven</td>
			<td>Boxun</td>
			<td>GZX-9140MBE</td>
			<td></td>
		</tr>
		<tr>
			<td>Isofluorane</td>
			<td>RWD Life Science</td>
			<td>902-0000-522</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereotaxic apparatus</td>
			<td>RWD Life Science</td>
			<td>900-0068-507</td>
			<td></td>
		</tr>
		<tr>
			<td>Anesthesia apparatus</td>
			<td>RWD Life Science</td>
			<td>902-0000-510</td>
			<td></td>
		</tr>
		<tr>
			<td>Homeothermic Heating Device</td>
			<td>Harvard Apparatus</td>
			<td>K 024509</td>
			<td></td>
		</tr>
		<tr>
			<td>Amplifier Model 3000&#160;</td>
			<td>A-M Systems</td>
			<td>61558</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro1401 Analog Digital converter</td>
			<td>Cambridge Electronic Design Ltd.</td>
			<td>4383</td>
			<td>Data acquisition unit</td>
		</tr>
		<tr>
			<td>Spike2</td>
			<td>Cambridge Electronic Design Ltd.</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

recording eeg in freely moving neonatal rats using a novel method

EEG is a useful method to detect electrical activity in the brain. Moreover, it is a widely used diagnostic tool for various neurological conditions, such as epilepsy and neurodegenerative disorders. However, it is technically difficult to obtain EEG recordings in neonates as it requires specialized handling and great care. Here, we present a novel method to record EEG in neonatal rat pups (P8-P15). We designed a simple and reliable electrode using computer pin loci; it can be easily implanted into the skull of a rat pup to record high-quality EEG signals in the normal and epileptic brain. Pups were given an intraperitoneal (i.p.) injection of the neurotoxin kainic acid (KA) to induce epileptic seizures. The surgical implantation performed in this procedure is less expensive than other EEG procedures for neonates. This method allows one to record high-quality and stable EEG signals for more than 1 week. Furthermore, this procedure can also be applied to adult rats and mice to study epilepsy or other neurological disorders.

If the above surgical procedures are conducted properly, one channel rat neonatal pup EEG recording will be successfully performed. 10 min after the KA injection, a regular pattern of behavioral signs emerged in the form of irregular movements and scratching, tremors, and loss of balance. Figure 2 shows the representative raw EEG traces and interictal, ictal-tonic, and ictal-clonic expanded traces. Recurrent interictal and ictal EEG discharge patterns started 15-60 min (30 &#177;5.2 min; mean &#177;SEM) after KA injection (Figure 2A and 2B). Figure 2D and 2E represents the tonic-clonic seizures that are the most common type. The ictal-tonic duration lasted 15.2 &#177;0.9 s (Figure 2D). The representative results showed that tonic discharges were followed by clonic bursts (Figure 2B, 2D, and 2E).
<img alt="Figure 1" src="/files/ftp_upload/55489/55489fig1.jpg" /> 
Figure 1: Illustration of Surgical Implantation Protocol. (A) Tiny computer pins. (B) Female pin (left) and male pin (right). The scale bar is used to measure the length of the female pin. (C) Connected female and male pin. (D) Exposed skull. No. 1 presents the reference site, No. 2 presents Bregma, No. 3 and 4 present recording sites, and No. 5 presents Lambda. (E) Electrode implantation. No. 1 presents the reference electrode, No. 3 and 4 present the recording electrode. (F) Recovered rat pup (P9). <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55489/55489fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/55489/55489fig2.jpg" /> 
Figure 2: KA-induced Neonatal Seizure. (A) A representative EEG trace from a P10 rat that was injected with KA (2 mg/kg, i.p.) showed recurrent epileptic burst discharges. (B) Extended view of single cluster-like epileptic discharges from the box in A. (C, D and E) Typical interictal, ictal-tonic, and ictal-clonic traces expanded from the box in B. (F) Summarized data showing the average EEG power (1 min time window) before and after the KA injection (mean &#177;SEM). <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55489/55489fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/55489/55489fig3.jpg" /> 
Figure 3: EEG Recording Set-up. Representative trace recording is shown in freely moving neonatal rat pup. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55489/55489fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Recording EEG in Freely Moving Neonatal Rats Using a Novel Method at JoVE.com

Recording EEG in Freely Moving Neonatal Rats Using a Novel Method

Here, we introduce a novel technique designed to record electroencephalography (EEG) in freely moving neonatal epileptic pups and describe its procedures, features, and applications. This method allows one to record EEG for more than 1 week.

EEG is a useful method to detect electrical activity in the brain. Moreover, it is a widely used diagnostic tool for various neurological conditions, such ...

recording-eeg-in-freely-moving-neonatal-rats-using-a-novel-method

Nanyang Technological University

Research

JoVE Journal

Neuroscience

13.1K Views.  South China Normal University. Here, we introduce a novel technique designed to record electroencephalography (EEG) in freely moving neonatal epileptic pups and describe its procedures, features, and applications. This method allows one to record EEG for more than 1 week.

Video: Recording EEG in Freely Moving Neonatal Rats Using a Novel Method

Novel Apparatus and Method for Drug Reinforcement

Animal models of reinforcement have proven to be useful for understanding the neurobiological mechanisms underlying drug addiction. Operant drug self-administration and conditioned place preference (CPP) procedures are expansively used in animal research to model various components of drug reinforcement, consumption, and addiction in humans. For this study, we used a novel approach to studying drug reinforcement in rats by combining traditional CPP and self-administration methodologies. We assembled an apparatus using two Med Associate operant chambers, sensory stimuli, and a Plexiglas-constructed neutral zone. These modifications allowed our experiments to encompass motivational aspects of drug intake through self-administration and drug-free assessment of drug/cue conditioning strength with the CPP test. In our experiments, rats self-administered cocaine (0.75 mg/kg/inj, i.v.) during either four (e.g., the "short-term") or eight (e.g., the "long-term") alternating-day sessions in an operant environment containing distinctive sensory cues (e.g., olfactory and visual). On the alternate days, in the other (differently-cued) operant environment, saline was available for self-infusion (0.1 ml, i.v.). Twenty-four hours after the last self-administration/cue-pairing session, a CPP test was conducted. Consistent with typical CPP findings, there was a significant preference for the chamber associated with cocaine self-administration. In addition, in animals undergoing the long-term experiment, a significant positive correlation between CPP magnitude and the number of cocaine-reinforced lever responses. In conclusion, this apparatus and approach is time and cost effective, can be used to examine a wide array of topics pertaining to drug abuse, and provides more flexibility in experimental design than CPP or self-administration methods alone.

Operant drug self-administration and conditioned place preference (CPP) procedures are expansively used in research to model various components of drug reinforcement, consumption, and addiction in humans. In this report, we combined traditional CPP and self-administration methods as a novel approach to studying drug reinforcement and addiction in rats.

Animal models of reinforcement have proven to be useful for understanding the neurobiological mechanisms underlying drug addiction.  Operant drug ...

High-density EEG Recordings of the Freely Moving Mice using Polyimide-based Microelectrode

Electroencephalogram (EEG) indicates the averaged electrical activity of the neuronal populations on a large-scale level. It is widely utilized as a noninvasive brain monitoring tool in cognitive neuroscience as well as a diagnostic tool for epilepsy and sleep disorders in neurology. However, the underlying mechanism of EEG rhythm generation is still under the veil. Recently introduced polyimide-based microelectrode (PBM-array) for high resolution mouse EEG1 is one of the trials to answer the neurophysiological questions on EEG signals based on a rich genetic resource that the mouse model contains for the analysis of complex EEG generation process. This application of nanofabricated PBM-array to mouse skull is an efficient tool for collecting large-scale brain activity of transgenic mice and accommodates to identify the neural correlates to certain EEG rhythms in conjunction with behavior. However its ultra-thin thickness and bifurcated structure cause a trouble in handling and implantation of PBM-array. In the presented video, the preparation and surgery steps for the implantation of PBM-array on a mouse skull are described step by step. Handling and surgery tips to help researchers succeed in implantation are also provided.

In this article, we described the surgery procedure and handling tips for implantation of ultra-thin polyimide-based microelectrode array (PBM-array) on the mouse skull for acquisition of high-density encephalography (EEG) in a mouse model.

Electroencephalogram (EEG) indicates the averaged electrical activity of the neuronal populations on a large-scale level. It is widely utilized as a ...

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 ...

Recording Single Neurons' Action Potentials from Freely Moving Pigeons Across Three Stages of Learning

While the subject of learning has attracted immense interest from both behavioral and neural scientists, only relatively few investigators have observed single-neuron activity while animals are acquiring an operantly conditioned response, or when that response is extinguished. But even in these cases, observation periods usually encompass only a single stage of learning, i.e. acquisition or extinction, but not both (exceptions include protocols employing reversal learning; see Bingman&#160;et al.1 for an example). However, acquisition and extinction entail different learning mechanisms and are therefore expected to be accompanied by different types and/or loci of neural plasticity.
Accordingly, we developed a behavioral paradigm which institutes three stages of learning in a single behavioral session and which is well suited for the simultaneous recording of single neurons&#39; action potentials. Animals are trained on a single-interval forced choice task which requires mapping each of two possible choice responses to the presentation of different novel visual stimuli (acquisition). After having reached a predefined performance criterion, one of the two choice responses is no longer reinforced (extinction). Following a certain decrement in performance level, correct responses are reinforced again (reacquisition). By using a new set of stimuli in every session, animals can undergo the acquisition-extinction-reacquisition process repeatedly. Because all three stages of learning occur in a single behavioral session, the paradigm is ideal for the simultaneous observation of the spiking output of multiple single neurons. We use pigeons as model systems, but the task can easily be adapted to any other species capable of conditioned discrimination learning.

Learning new stimulus-response associations engages a wide range of neural processes which are ultimately reflected in changing spike output of individual neurons. Here we describe a behavioral protocol allowing for the continuous registration of single-neuron activity while animals acquire, extinguish, and reacquire a conditioned response within a single experimental session.

While the subject of learning has attracted immense interest from both behavioral and neural scientists, only relatively few investigators have observed ...

Extracellular Wire Tetrode Recording in Brain of Freely Walking Insects

Increasing interest in the role of brain activity in insect motor control requires that we be able to monitor neural activity while insects perform natural behavior. We previously developed a technique for implanting tetrode wires into the central complex of cockroach brains that allowed us to record activity from multiple neurons simultaneously while a tethered cockroach turned or altered walking speed. While a major advance, tethered preparations provide access to limited behaviors and often lack feedback processes that occur in freely moving animals. We now present a modified version of that technique that allows us to record from the central complex of freely moving cockroaches as they walk in an arena and deal with barriers by turning, climbing or tunneling. Coupled with high speed video and cluster cutting, we can now relate brain activity to various parameters of the movement of freely behaving insects.

We previously developed a technique for implanting tetrode wires into the central complex of cockroach brains that allows us to monitor activity in individual units of tethered cockroaches. Here we present a modified version of that technique that allows us to also record brain activity in freely moving insects.

Increasing interest in the role of brain activity in insect motor control requires that we be able to monitor neural activity while insects perform ...

A Novel Behavioral Assay to Investigate Gustatory Responses of Individual, Freely-moving Bumble Bees (Bombus terrestris)

Generalist pollinators like the buff-tailed bumble bee, Bombus terrestris, encounter both nutrients and toxins in the floral nectar they collect from flowering plants. Only a few studies have described the gustatory responses of bees toward toxins in food, and these experiments have mainly used the proboscis extension response on restrained honey bees. Here, a new behavioral assay is presented for measuring the feeding responses of freely-moving, individual worker bumble bees to nutrients and toxins. This assay measures the amount of solution ingested by each bumble bee and identifies how tastants in food influence the microstructure of the feeding behavior.
The solutions are presented in a microcapillary tube to individual bumble bees that have been previously starved for 2-4 hr. The behavior is captured on digital video. The fine structure of the feeding behavior is analyzed by continuously scoring the position of the proboscis (mouthparts) from video recordings using event logging software. The position of the proboscis is defined by three different behavioral categories: (1) proboscis is extended and in contact with the solution, (2) proboscis is extended but not in contact with the solution and (3) proboscis is stowed under the head. Furthermore the speed of the proboscis retracting away from the solution is also estimated.
In the present assay the volume of solution consumed, the number of feeding bouts, the duration of the feeding bouts and the speed of the proboscis retraction after the first contact is used to evaluate the phagostimulatory or the deterrent activity of the compounds tested.
This new taste assay will allow researchers to measure how compounds found in nectar influence the feeding behavior of bees and will also be useful to pollination biologists, toxicologists and neuroethologists studying the bumble bee&#39;s taste system.

A Novel Behavioral Assay to Investigate Gustatory Responses of Individual, Freely-moving Bumble Bees Bombus terrestris

A novel behavioral assay is described for investigating the short term gustatory responses of the mouthparts of freely-moving bumble bees (Bombus terrestris) toward nutrients and toxins in solution.

Generalist pollinators like the buff-tailed bumble bee, Bombus terrestris, encounter both nutrients and toxins in the floral nectar they collect from ...

Noninvasive EEG Recordings from Freely Moving Piglets

The method allows the recording of high-quality electroencephalograms (EEGs) from freely moving piglets directly in the pigpen. We use a one-channel telemetric electroencephalography system in combination with standard self-adhesive hydrogel electrodes. The piglets are calmed down without the use of sedatives. After their release into the pigpen, the piglets behave normally&#8212;they drink and sleep in the same cycle as their siblings. Their sleep phases are used for the EEG recordings.

Here, we present a protocol to record telemetric electroencephalograms (EEGs) from freely moving piglets directly in the pigpen without the use of a sedative, making it possible to record typical EEG patterns during non-REM sleep, like spindle bursts.

The method allows the recording of high-quality electroencephalograms (EEGs) from freely moving piglets directly in the pigpen. We use a one-channel ...

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 ...

Hybrid Microdrive System with Recoverable Opto-Silicon Probe and Tetrode for Dual-Site High Density Recording in Freely Moving Mice

Multi-regional neural recordings can provide crucial information to understanding fine-timescale interactions between multiple brain regions. However, conventional microdrive designs often only allow use of one type of electrode to record from single or multiple regions, limiting the yield of single-unit or depth profile recordings. It also often limits the ability to combine electrode recordings with optogenetic tools to target pathway and/or cell type specific activity. Presented here is a hybrid microdrive array for freely moving mice to optimize yield and a description of its fabrication and reuse of the microdrive array. The current design employs nine tetrodes and one opto-silicon probe implanted in two different brain areas simultaneously in freely moving mice. The tetrodes and the opto-silicon probe are independently adjustable along the dorsoventral axis in the brain to maximize the yield of unit and oscillatory activities. This microdrive array also incorporates a set-up for light, mediating optogenetic manipulation to investigate the regional- or cell type-specific responses and functions of long-range neural circuits. In addition, the opto-silicon probe can be safely recovered and reused after each experiment. Because the microdrive array consists of 3D-printed parts, the design of microdrives can be easily modified to accommodate various settings. First described is the design of the microdrive array and how to attach the optical fiber to a silicon probe for optogenetics experiments, followed by fabrication of the tetrode bundle and implantation of the array into a mouse brain. The recording of local field potentials and unit spiking combined with optogenetic stimulation also demonstrate feasibility of the microdrive array system in freely moving mice.

This protocol describes the construction of a hybrid microdrive array that allows implantation of nine independently adjustable tetrodes and one adjustable opto-silicon probe in two brain regions in freely moving mice. Also demonstrated is a method for safely recovering and reusing the opto-silicon probe for multiple purposes.

Multi-regional neural recordings can provide crucial information to understanding fine-timescale interactions between multiple brain regions. However, ...

Multi-Fiber Photometry to Record Neural Activity in Freely-Moving Animals

Recording the activity of a group of neurons in a freely-moving animal is a challenging undertaking. Moreover, as the brain is dissected into smaller and smaller functional subgroups, it becomes paramount to record from projections and/or genetically-defined subpopulations of neurons. Fiber photometry is an accessible and powerful approach that can overcome these challenges. By combining optical and genetic methodologies, neural activity can be measured in deep brain structures by expressing genetically-encoded calcium indicators, which translate neural activity into an optical signal that can be easily measured. The current protocol details the components of a multi-fiber photometry system, how to access deep brain structures to deliver and collect light, a method to account for motion artifacts, and how to process and analyze fluorescent signals. The protocol details experimental considerations when performing single and dual color imaging, from either single or multiple implanted optic fibers.

This protocol details how to implement and perform multi-fiber photometry recordings, how to correct for calcium-independent artifacts, and important considerations for dual-color photometry imaging.

Recording the activity of a group of neurons in a freely-moving animal is a challenging undertaking. Moreover, as the brain is dissected into smaller and ...