Name: operant protocols for assessing the cost-benefit analysis during reinforced decision making by rodents
Uploaded: 2018-09-10T13:30:00
Description: Watch this Scientific Journal Video about Operant Protocols for Assessing the Cost-benefit Analysis During Reinforced Decision Making by Rodents at JoVE.com

This research was supported by RMH Neuroscience Foundation, Australia; the Australian Brain Foundation; the RACP Thyne Reid Fellowship, Australia; and by a project grant from the Cognitive Sciences and Technologies Council, Iran to Abbas Haghparast.

All procedures explained here were approved and carried out in accordance with the Guide for the Care and Use of Laboratory Animals and were approved by the Florey Institute Animal Ethics Committee or the Neuroscience Research Center.
1. Housing, Handling, and Food Restriction
<ol>
	<li>Use adult (normally 8 weeks old) male rats (any strains) and keep them in the room with a 12-h light/dark cycle.</li>
	<li>Restrict their food access to encourage the animals to perform the task. 
	Note:...

<ol>
	<li>Gold, J. I., Shadlen, M. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+neural+basis+of+decision+making.">The neural basis of decision making.</a> Annual Review of Neuroscience. 30, 535-574 (2007).</li><li>Shi, Z., M&#252;ller, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multisensory+perception+and+action:+development,+decision-making,+and+neural+mechanisms.">Multisensory perception and action: development, decision-making, and neural mechanisms.</a> Frontiers in Integrative Neuroscience. 7, 81 (2013).</li><li>Sutton, R. S., Barto, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Reinforcement Learning: An Introduction. 1, (1998).</li><li>Khani, A., Rainer, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+and+neurochemical+basis+of+reinforcement-guided+decision+making.">Neural and neurochemical basis of reinforcement-guided decision making.</a> Journal of Neurophysiology. 116, 724-741 (2016).</li><li>Fatahi, Z., Haghparast, A., Khani, A., Kermani, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+connectivity+between+anterior+cingulate+cortex+and+orbitofrontal+cortex+during+value-based+decision+making.">Functional connectivity between anterior cingulate cortex and orbitofrontal cortex during value-based decision making.</a> Neurobiology of Learning and Memory. 147, 74-78 (2018).</li><li>Khani, 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=Activation+of+cannabinoid+system+in+anterior+cingulate+cortex+and+orbitofrontal+cortex+modulates+cost-benefit+decision+making.">Activation of cannabinoid system in anterior cingulate cortex and orbitofrontal cortex modulates cost-benefit decision making.</a> Psychopharmacology. 232, 2097-2112 (2015).</li><li>Rudebeck, P. H., Walton, M. E., Smyth, A. N., Bannerman, D. M., Rushworth, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Separate+neural+pathways+process+different+decision+costs.">Separate neural pathways process different decision costs.</a> Nature Neuroscience. 9, 1161-1168 (2006).</li><li>Gage, G. J., 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=Surgical+implantation+of+chronic+neural+electrodes+for+recording+single+unit+activity+and+electrocorticographic+signals.">Surgical implantation of chronic neural electrodes for recording single unit activity and electrocorticographic signals.</a> Journal of Visualized Experiments. (60), e3565 (2012).</li><li>Paxinos, G., Watson, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Rat Brain in Stereotaxic Coordinates. , (1998).</li><li>Bokil, H., Andrews, P., Kulkarni, J. E., Mehta, S., Mitra, P. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronux:+a+platform+for+analyzing+neural+signals.">Chronux: a platform for analyzing neural signals.</a> Journal of Neuroscience Methods. 192, 146-151 (2010).</li><li>Cohen, M. X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Analyzing Neural Time Series Data: Theory and Practice. , (2014).</li><li>Luk, C. -. H., Wallis, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Choice+coding+in+frontal+cortex+during+stimulus-guided+or+action-guided+decision-making.">Choice coding in frontal cortex during stimulus-guided or action-guided decision-making.</a> Journal of Neuroscience. 33, 1864-1871 (2013).</li><li>Rudebeck, P. H., 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=Frontal+cortex+subregions+play+distinct+roles+in+choices+between+actions+and+stimuli.">Frontal cortex subregions play distinct roles in choices between actions and stimuli.</a> Journal of Neuroscience. 28, 13775-13785 (2008).</li><li>Goshadrou, F., Kermani, M., Ronaghi, A., Sajjadi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+ghrelin+on+MK-801+induced+memory+impairment+in+rats.">The effect of ghrelin on MK-801 induced memory impairment in rats.</a> Peptides. 44, 60-65 (2013).</li><li>Haghparast, 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=Intrahippocampal+administration+of+D2+but+not+D1+dopamine+receptor+antagonist+suppresses+the+expression+of+conditioned+place+preference+induced+by+morphine+in+the+ventral+tegmental+area.">Intrahippocampal administration of D2 but not D1 dopamine receptor antagonist suppresses the expression of conditioned place preference induced by morphine in the ventral tegmental area.</a> Neuroscience Letters. 541, 138-143 (2013).</li><li>Esmaeili, M. -. H., Kermani, M., Parvishan, A., Haghparast, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+D1/D2+dopamine+receptors+in+the+CA1+region+of+the+rat+hippocampus+in+the+rewarding+effects+of+morphine+administered+into+the+ventral+tegmental+area.">Role of D1/D2 dopamine receptors in the CA1 region of the rat hippocampus in the rewarding effects of morphine administered into the ventral tegmental area.</a> Behavioural Brain Research. 231, 111-115 (2012).</li><li>Chaleek, N., Kermani, M., Eliassi, A., Haghparast, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+orexin+and+glucose+microinjected+into+the+hypothalamic+paraventricular+nucleus+on+gastric+acid+secretion+in+conscious+rats.">Effects of orexin and glucose microinjected into the hypothalamic paraventricular nucleus on gastric acid secretion in conscious rats.</a> Neurogastroenterology &amp; Motility. 24, e94-e102 (2012).</li><li>Kermani, M., Eliassi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gastric+acid+secretion+induced+by+paraventricular+nucleus+microinjection+of+orexin+A+is+mediated+through+activation+of+neuropeptide+Yergic+system.">Gastric acid secretion induced by paraventricular nucleus microinjection of orexin A is mediated through activation of neuropeptide Yergic system.</a> Neuroscience. 226, 81-88 (2012).</li><li>Kermani, M., Azizi, P., Haghparast, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+nitric+oxide+in+the+effects+of+cumin+(Cuminum+Cyminum+L.)+fruit+essential+oil+on+the+acquisition+of+morphine-induced+conditioned+place+preference+in+adult+male+mice.">The role of nitric oxide in the effects of cumin (Cuminum Cyminum L.) fruit essential oil on the acquisition of morphine-induced conditioned place preference in adult male mice.</a> Chinese Journal of Integrative Medicine. , 1-6 (2012).</li><li>Ahmadi, 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=Synthesis+and+antinociceptive+behaviors+of+new+methyl+and+hydroxyl+derivatives+of+phencyclidine.">Synthesis and antinociceptive behaviors of new methyl and hydroxyl derivatives of phencyclidine.</a> Current Medicinal Chemistry. 19, 763-769 (2012).</li></ol>

Rodents have long been used in neuroscientific studies that deal with different topics, from cognitive abilities such as learning and memory2,14 and reinforced behavior7,15,16 to the central control of organs17,18 and neuropharmacology19,20. The proposed ...

Decision making is the process of recognizing and selecting choices based on the values and preferences of the decision maker and the consequences of the selected action1. Although decision making has been extensively studied in different fields (i.e., economics, psychology, and neuroscience), neural mechanisms underlying such cognitive abilities are not yet fully understood. Two subcategories of decision making are perceptual decision making and reinforcement-guided decision making. Though they incorporate considerable overlapping elements and concepts, perceptual decision making relies on the available sensory information1,2, whereas reinforcement-guided decision-making deals with the relative value of actions gained over a specific timescale3. One important aspect of reinforced decision making is the cost-benefit analysis which is performed intuitively by the brain by computing the benefits of the given choices and subtracting the associated costs of each alternative1.
The T-maze (or the variant Y-maze) is one of the most-used mazes in cognitive experiments using rodents. Animals are placed in the start arm (the base of the T) and permitted to choose the goal arm (one of the side arms). Tasks such as a forced alternation or left-right discrimination are mainly used with rodents in the T-maze to test reference and working memory4. T-mazes are also widely used in decision-making experiments5,6,7. In the simplest design, the reward is placed in only one goal arm. The choice is predictable, and animals would certainly prefer the reward rather than nothing, regardless of the reward value. Another option is to place rewards in both goal arms and then let the animals make a choice of which path to take depending on several parameters (i.e., the natural preference of the animal, the difference in the value of the rewards, and the costs to be paid). In the value-based design, the task is more complicated by having weighing-scale properties. In this way, an animal receives differently valued rewards by choosing between the two alternatives, as well as between the costs of the actions [i.e., the amount of waiting (delay-based) or the amount of effort (effort-based) needed to receive rewards], each contributing to the decision that is made5,6.
In traditional delay-based T-maze decision making, animals are trained to select the high reward arm (HRA) and avoid the opposite low reward arm (LRA). The sides of the HRA and the LRA remain unchanged throughout the experiment. Although the task described above has been well documented in the literature, it suffers from several procedural drawbacks. Firstly, by having a fixed goal arm, the animal knows which arm to choose from the beginning of each trial. In this scenario, animals may select the goal arm based on their memory rather than on decision making. Hence, in a delay-based decision-making paradigm, if an animal selects the low reward because of the study intervention, it will not be clear whether this is due to a loss of memory or to the study intervention. A memory control group to segregate the observed behavior from the memory problem might be considered, but this burdens researchers and animals alike because of the additional work7. A second concern is the moment of decision making by the animal: once animals reach the decision zone (the junction of all three arms), they usually look to the left and to the right, weigh the costs and benefits regarding each arm, and then make their decision. However, after a few trials, they perform such a computation prior to arriving at the decision zone and simply run directly to the reward arm. As a result, these two drawbacks&#8212;a pre-bias to one arm and finding the moment of decision making&#8212;both highly interrupt the interpretation of electrophysiological and neuroimaging data.
In the method explained in this paper, the preferred arm (HRA) is cued by an auditory cue and may vary from trial to trial. Animals initiate the trials by entering the test zone (Figure 1) and triggering the auditory cue by &#34;nose-poking&#34; an infrared gate that has been placed at the junction of the three arms. The audio signal (20 dB, between 500 and 1,000 ms) is played from a speaker at the end of the goal arm.

<table>
	<tbody>
		<tr>
			<td>T-maze</td>
			<td></td>
			<td></td>
			<td>Self made</td>
		</tr>
		<tr>
			<td>Dustless Precision Sugar Pellets</td>
			<td>TSE Systems Intl. Group</td>
			<td>F0023</td>
			<td>45 mg, Sucrose</td>
		</tr>
		<tr>
			<td>Ketamine Hydrochloride Injection, USP</td>
			<td>Sigma-Aldrich</td>
			<td>6740-87-0</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylazine</td>
			<td>Sigma-Aldrich</td>
			<td>7361-61-7</td>
			<td></td>
		</tr>
		<tr>
			<td>stereotaxic device</td>
			<td>Stoelting</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Isofluran</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-363629Rx</td>
			<td></td>
		</tr>
		<tr>
			<td>PFA-coated stainless-steel wires</td>
			<td>A-M systems</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>acrylic cement</td>
			<td>Vertex, MA, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>(wooden or PVC (polyvinyl chloride)-made)</td>
			<td>local suppliers</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Mini-Fit Power Connector</td>
			<td>Molex</td>
			<td>15243048</td>
			<td></td>
		</tr>
		<tr>
			<td>ethannol 70%</td>
			<td>Local suppliers</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>buprenorphine</td>
			<td>diamondback drugs</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Arduino UNO</td>
			<td>Arduino</td>
			<td></td>
			<td>https://www.arduino.cc/</td>
		</tr>
		<tr>
			<td>Infrared emitting diode</td>
			<td>Sharp</td>
			<td>GL480E00000F</td>
			<td>http://www.sharp-world.com/</td>
		</tr>
		<tr>
			<td>Chronux Toolbox</td>
			<td></td>
			<td></td>
			<td>Chronux.org</td>
		</tr>
		<tr>
			<td>Arduino codes</td>
			<td></td>
			<td></td>
			<td>https://github.com/dechuans/arduino-maze</td>
		</tr>
	</tbody>
</table>

operant protocols for assessing the cost-benefit analysis during reinforced decision making by rodents

Reinforcement-guided decision making is the ability to choose between competing courses of action based on the relative value of the benefits and their consequences. This process is integral to the normal human behavior and has been shown to be disrupted by neurological and psychiatric disorders such as addiction, schizophrenia, and depression. Rodents have long been used to uncover the neurobiology of human cognition. To this end, several behavioral tasks have been developed; however, most are non-automated and are labor-intensive. The recent development of the open-source microcontroller has enabled researchers to automate operant-based tasks for assessing a variety of cognitive tasks, standardizing the stimulus presentation, improving the data recording and consequently, improving the research output. Here, we describe an automated delay-based reinforcement-guided decision-making task, using an operant T-maze controlled by custom-written software programs. Using these decision-making tasks, we show the changes in the local field potential activities in the anterior cingulate cortex of a rat whilst it performs a delay-based cost-and-benefit decision-making task.

The data presented here is the recorded LFP from the left orbitofrontal cortex (OFC) and the anterior cingulate cortex (ACC) of six male Wistar rats using bipolar electrodes (of PFA-coated stainless steel). Table 1 shows the behavioral acquisition length for each training stage. The coordinates for the target locations were determined from a rat brain atlas9 and are as follows: for the AAC, 1.2 mm anterior to the bregma, 0.8 mm lateral to the midli...

Watch this Scientific Journal Video about Operant Protocols for Assessing the Cost-benefit Analysis During Reinforced Decision Making by Rodents at JoVE.com

Operant Protocols for Assessing the Cost-benefit Analysis During Reinforced Decision Making by Rodents

A cost-benefit analysis is a weighing-scale approach that the brain performs during the course of decision making. Here, we propose a protocol to train rats on an operant-based decision-making paradigm where rats choose higher rewards at the expense of waiting for 15 s to receive them.

Reinforcement-guided decision making is the ability to choose between competing courses of action based on the relative value of the benefits and their ...

This technique can answer key questions in cognitive neurosciences such as how comparing consequences and causes can affect making a decision. The main advantage of this technique is that the special elements such as the location of the board do not affect the animal's decision. Begin by restricting food access to adult male rats in a room with a 12-hour light/dark cycle.On days one to three, handle the animals for five minutes twice a day. Then, weigh the animals after each handling session to determine the free-feeding amount. Next, obtain a T maze equipped with three partitions or arms, two stimulus speakers, five retractable doors, and five infrared motion sensors or Infrared Beam or IRB sensors.Copy the experimental software code into the computer. Then, download the experimental software and install it on a computer. Connect the microcontroller to the computer using a USB port.Next, on the computer, click the software icon, go to Tools and then select Port. From the dropdown menu, choose the COM port which connects the software to the computer. Go to Tools and select Boards.From the dropdown menu, select the type of software that controls the T maze. Then, click Upload on the top left of the interface window. Select the experimental software code, wait until the process finishes.After the process has finished, click Serial Monitor at the top right of the interface window. Then, in a new popup window, change the baud rate to 115, 200. For five days prior to the experimental day, bring the animals to the experiment room for at least one hour.Leave 10 sugar pellets at each goal arm and leave all doors open. During days one through three, place the animals individually in the maze for periods of 10 minutes once per day to explore the maze with no restrictions. During days four through five, place the animals individually in the maze.Leave two pellets in each arm and allow them to sample the food from both sides. Remove the animal from the maze immediately after eating the pellets from both sides or after five minutes of being in the maze. Finally, after each habituation session, clean the maze's floors with 70%ethanol and make sure the ethanol has evaporated before placing the next animal in the maze.On day six, bring the animals to the experiment room at least one hour prior to the experiment. Run the software program and set the trial number to 14 of which the first four trials will be forced choice and the rest will be choice trials. Before each trial begins, place four pellets in the goal arm which is to be cued as the High Reward Arm, HRA, and two pellets in the other arm which is the Low Reward Arm, LRA.For the forced choice trial training, pseudo randomly block one arm before each trial so that the animal is forced to select the other arm. Place an animal in the start box. After five to seven seconds, simultaneously open the start gate and click Start in the software's interface window.Collect the animal immediately after eating pellets or after five minutes of being in the maze and leave the animal in the home cage for two minutes. Prior to each trial, close door B in the arm which is selected by the software to be the HRA. Leave door A open in the opposite arm, the LRA.Then, place an animal in the start box. After five to seven seconds, simultaneously open the start gate and click Start. Let the animal freely choose either arm.If the animal chooses the HRA, open door A, let the animal enter the chamber, shut door A and open door B immediately to give the animal access to the selected food well. If the animal chooses the LRA, open door B in order to give the animal access to the food well. Next, remove the animal after it has eaten all the food in the selected food well and leave it in the home cage for two minutes.Complete the 10 choice trials for each animal and record the animal's choice, either HRA or LRA in each trial. Calculate the percentage of High Reward Choice, HRC, for all choice trials after each training session. Finally, after each animal has reached 80%of HRC in the discrimination training, complete the delay training by opening door B after a five-second delay once the animal chooses the HRA.Run 10 trials per day for each animal. Results indicated that there was a decrease in both the low and the high frequency powers in the ACC from the onset to the end of the stimulus. Comparing the time spent outside the chamber with the time in the chamber, the spectral analysis showed no changes in the oscillatory activities in the ACC.Further, mid to high frequency band activities in the OFC increased following the stimulus onset. No significant changes were observed in the pre-chamber and chamber time windows when they were compared together. While attempting this procedure, it is important to remove any source of anxiety as it can affect the animal's decision and impair the reliability of the result.

operant-protocols-for-assessing-cost-benefit-analysis-during

Research

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Neuroscience

Combining Behavioral Endocrinology and Experimental Economics: Testosterone and Social Decision Making

Behavioral endocrinological research in humans as well as in animals suggests that testosterone plays a key role in social interactions. Studies in rodents have shown a direct link between testosterone and aggressive behavior1 and folk wisdom adapts these findings to humans, suggesting that testosterone induces antisocial, egoistic or even aggressive behavior2. However, many researchers doubt a direct testosterone-aggression link in humans, arguing instead that testosterone is primarily involved in status-related behavior3,4. As a high status can also be achieved by aggressive and antisocial means it can be difficult to distinguish between anti-social and status seeking behavior.
We therefore set up an experimental environment, in which status can only be achieved by prosocial means. In a double-blind and placebo-controlled experiment, we administered a single sublingual dose of 0.5 mg of testosterone (with a hydroxypropyl-&beta;-cyclodextrin carrier) to 121 women and investigated their social interaction behavior in an economic bargaining paradigm. Real monetary incentives are at stake in this paradigm; every player A receives a certain amount of money and has to make an offer to another player B on how to share the money. If B accepts, she gets what was offered and player A keeps the rest. If B refuses the offer, nobody gets anything. A status seeking player A is expected to avoid being rejected by behaving in a prosocial way, i.e. by making higher offers.
The results show that if expectations about the hormone are controlled for, testosterone administration leads to a significant increase in fair bargaining offers compared to placebo. The role of expectations is reflected in the fact that subjects who report that they believe to have received testosterone make lower offers than those who say they believe that they were treated with a placebo. These findings suggest that the experimental economics approach is sensitive for detecting neurobiological effects as subtle as those achieved by administration of hormones. Moreover, the findings point towards the importance of both psychosocial as well as neuroendocrine factors in determining the influence of testosterone on human social behavior.

The procedure described in this protocol shows that testosterone administration and folk beliefs about testosterone may be associated with directly opposed social behaviors.

Behavioral endocrinological research in humans as well as in animals suggests that testosterone plays a key role in social interactions. Studies in ...

Operant Sensation Seeking in the Mouse

Operant methods are powerful behavioral tools for the study of motivated behavior. These 'self-administration' methods have been used extensively in drug addiction research due to their high construct validity. Operant studies provide researchers a tool for preclinical investigation of several aspects of the addiction process. For example, mechanisms of acute reinforcement (both drug and non-drug) can be tested using pharmacological or genetic tools to determine the ability of a molecular target to influence self-administration behavior1-6. Additionally, drug or food seeking behaviors can be studied in the absence of the primary reinforcer, and the ability of pharmacological compounds to disrupt this process is a preclinical model for discovery of molecular targets and compounds that may be useful for the treatment of addiction3,7-9. One problem with performing intravenous drug self-administration studies in the mouse is the technical difficulty of maintaining catheter patency. Attrition rates in these experiments are high and can reach 40% or higher10-15. Another general problem with drug self-administration is discerning which pharmacologically-induced effects of the reinforcer produce specific behaviors. For example, measurement of the reinforcing and neurological effects of psychostimulants can be confounded by their psychomotor effects. Operant methods using food reinforcement can avoid these pitfalls, although their utility in studying drug addiction is limited by the fact that some manipulations that alter drug self-administration have a minimal impact on food self-administration. For example, mesolimbic dopamine lesion or knockout of the D1 dopamine receptor reduce cocaine self-administration without having a significant impact on food self-administration 12,16. 
Sensory stimuli have been described for their ability to support operant responding as primary reinforcers (i.e. not conditioned reinforcers)17-22. Auditory and visual stimuli are self-administered by several species18,21,23, although surprisingly little is known about the neural mechanisms underlying this reinforcement. The operant sensation seeking (OSS) model is a robust model for obtaining sensory self-administration in the mouse, allowing the study of neural mechanisms important in sensory reinforcement24. An additional advantage of OSS is the ability to screen mutant mice for differences in operant behavior that may be relevant to addiction. We have reported that dopamine D1 receptor knockout mice, previously shown to be deficient in psychostimulant self-administration, also fail to acquire OSS24. This is a unique finding in that these mice are capable of learning an operant task when food is used as a reinforcer. While operant studies using food reinforcement can be useful in the study of general motivated behavior and the mechanisms underlying food reinforcement, as mentioned above, these studies are limited in their application to studying molecular mechanisms of drug addiction. Thus, there may be similar neural substrates mediating sensory and psychostimulant reinforcement that are distinct from food reinforcement, which would make OSS a particularly attractive model for the study of drug addiction processes. The degree of overlap between other molecular targets of OSS and drug reinforcers is unclear, but is a topic that we are currently pursuing. While some aspects of addiction such as resistance to extinction may be observed with OSS, we have found that escalation 25 is not observed in this model24. Interestingly, escalation of intake and some other aspects of addiction are observed with self-administration of sucrose26. Thus, when non-drug operant procedures are desired to study addiction-related processes, food or sensory reinforcers can be chosen to best fit the particular question being asked. 
In conclusion, both food self-administration and OSS in the mouse have the advantage of not requiring an intravenous catheter, which allows a higher throughput means to study the effects of pharmacological or genetic manipulation of neural targets involved in motivation. While operant testing using food as a reinforcer is particularly useful in the study of the regulation of food intake, OSS is particularly apt for studying reinforcement mechanisms of sensory stimuli and may have broad applicability to novelty seeking and addiction.

In this protocol we describe a method of operant learning using sensory stimuli as a reinforcer in the mouse. It requires no prior training or food restriction, and it allows the study of motivated behavior without the use of a pharmacological or natural reinforcer such as food.

Operant methods are powerful behavioral tools for the study of motivated behavior.  These 'self-administration' methods have been used extensively in drug ...

Isolating Nasal Olfactory Stem Cells from Rodents or Humans

The olfactory mucosa, located in the nasal cavity, is in charge of detecting odours. It is also the only nervous tissue that is exposed to the external environment and easily accessible in every living individual. As a result, this tissue is unique for anyone aiming to identify molecular anomalies in the pathological brain or isolate adult stem cells for cell therapy. 
Molecular abnormalities in brain diseases are often studied using nervous tissue samples collected post-mortem. However, this material has numerous limitations. In contrast, the olfactory mucosa is readily accessible and can be biopsied safely without any loss of sense of smell1. Accordingly, the olfactory mucosa provides an "open window" in the adult human through which one can study developmental (e.g. autism, schizophrenia)2-4 or neurodegenerative (e.g. Parkinson, Alzheimer) diseases4,5. Olfactory mucosa can be used for either comparative molecular studies4,6 or in vitro experiments on neurogenesis3,7.
The olfactory epithelium is also a nervous tissue that produces new neurons every day to replace those that are damaged by pollution, bacterial of viral infections. This permanent neurogenesis is sustained by progenitors but also stem cells residing within both compartments of the mucosa, namely the neuroepithelium and the underlying lamina propria8-10. We recently developed a method to purify the adult stem cells located in the lamina propria and, after having demonstrated that they are closely related to bone marrow mesenchymal stem cells (BM-MSC), we named them olfactory ecto-mesenchymal stem cells (OE-MSC)11.
Interestingly, when compared to BM-MSCs, OE-MSCs display a high proliferation rate, an elevated clonogenicity and an inclination to differentiate into neural cells. We took advantage of these characteristics to perform studies dedicated to unveil new candidate genes in schizophrenia and Parkinson's disease4. We and others have also shown that OE-MSCs are promising candidates for cell therapy, after a spinal cord trauma12,13, a cochlear damage14 or in an animal models of Parkinson's disease15 or amnesia16.
In this study, we present methods to biopsy olfactory mucosa in rats and humans. After collection, the lamina propria is enzymatically separated from the epithelium and stem cells are purified using an enzymatic or a non-enzymatic method. Purified olfactory stem cells can then be either grown in large numbers and banked in liquid nitrogen or induced to form spheres or differentiated into neural cells. These stem cells can also be used for comparative omics (genomic, transcriptomic, epigenomic, proteomic) studies.

We describe here a method for biopsying olfactory mucosa from rat and human nasal cavities. These biopsies can be used for either identifying molecular anomalies in brain diseases or isolating multipotent adult stem cells that can be utilized for cell transplantation in animal models of brain trauma/disease.

The olfactory mucosa, located in the nasal cavity, is in charge of detecting odours. It is also the only nervous tissue that is exposed to the external ...

T-maze Forced Alternation and Left-right Discrimination Tasks for Assessing Working and Reference Memory in Mice

Forced alternation and left-right discrimination tasks using the T-maze have been widely used to assess working and reference memory, respectively, in rodents. In our laboratory, we evaluated the two types of memory in more than 30 strains of genetically engineered mice using the automated version of this apparatus. Here, we present the modified T-maze apparatus operated by a computer with a video-tracking system and our protocols in a movie format. The T-maze apparatus consists of runways partitioned off by sliding doors that can automatically open downward, each with a start box, a T-shaped alley, two boxes with automatic pellet dispensers at one side of the box, and two L-shaped alleys. Each L-shaped alley is connected to the start box so that mice can return to the start box, which excludes the effects of experimenter handling on mouse behavior. This apparatus also has an advantage that in vivo microdialysis, in vivo electrophysiology, and optogenetics techniques can be performed during T-maze performance because the doors are designed to go down into the floor. In this movie article, we describe T-maze tasks using the automated apparatus and the T-maze performance of &alpha;-CaMKII+/- mice, which are reported to show working memory deficits in the eight-arm radial maze task. Our data indicated that &alpha;-CaMKII+/- mice showed a working memory deficit, but no impairment of reference memory, and are consistent with previous findings using the eight-arm radial maze task, which supports the validity of our protocol. In addition, our data indicate that mutants tended to exhibit reversal learning deficits, suggesting that &alpha;-CaMKII deficiency causes reduced behavioral flexibility. Thus, the T-maze test using the modified automatic apparatus is useful for assessing working and reference memory and behavioral flexibility in mice.

This article presents the protocol of T-maze tests using a modified automated apparatus for assessing the learning and memory functions in mice.

Forced alternation and left-right discrimination tasks using the T-maze have been widely used to assess working and reference memory, respectively, in ...

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

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

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

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

A Fully Automated and Highly Versatile System for Testing Multi-cognitive Functions and Recording Neuronal Activities in Rodents

We have developed a fully automated system for operant behavior testing and neuronal activity recording by which multiple cognitive brain functions can be investigated in a single task sequence. The unique feature of this system is a custom-made, acoustically transparent chamber that eliminates many of the issues associated with auditory cue control in most commercially available chambers. The ease with which operant devices can be added or replaced makes this system quite versatile, allowing for the implementation of a variety of auditory, visual, and olfactory behavioral tasks. Automation of the system allows fine temporal (10 ms) control and precise time-stamping of each event in a predesigned behavioral sequence. When combined with a multi-channel electrophysiology recording system, multiple cognitive brain functions, such as motivation, attention, decision-making, patience, and rewards, can be examined sequentially or independently.

In this report, we present a fully automated and highly versatile system capable of simultaneously testing multi-cognitive behaviors and recording neuronal activities for rodents.

We have developed a fully automated system for operant behavior testing and neuronal activity recording by which multiple cognitive brain functions can be ...

A Polished and Reinforced Thinned-skull Window for Long-term Imaging of the Mouse Brain

In vivo imaging of cortical function requires optical access to the brain without disruption of the intracranial environment. We present a method to form a polished and reinforced thinned skull (PoRTS) window in the mouse skull that spans several millimeters in diameter and is stable for months. The skull is thinned to 10 to 15 &mu;m in thickness with a hand held drill to achieve optical clarity, and is then overlaid with cyanoacrylate glue and a cover glass to: 1) provide rigidity, 2) inhibit bone regrowth and 3) reduce light scattering from irregularities on the bone surface. Since the skull is not breached, any inflammation that could affect the process being studied is greatly reduced. Imaging depths of up to 250 &mu;m below the cortical surface can be achieved using two-photon laser scanning microscopy. This window is well suited to study cerebral blood flow and cellular function in both anesthetized and awake preparations. It further offers the opportunity to manipulate cell activity using optogenetics or to disrupt blood flow in targeted vessels by irradiation of circulating photosensitizers.

We present a method to form an imaging window in the mouse skull that spans millimeters and is stable for months without inflammation of the brain. This method is well suited for longitudinal studies of blood flow, cellular dynamics, and cell/vascular structure using two-photon microscopy.

In vivo imaging of cortical function requires optical access to the brain without disruption of the intracranial environment. We present a method to form ...

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.  ...

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

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

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

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

Effects of Transcranial Alternating Current Stimulation on the Primary Motor Cortex by Online Combined Approach with Transcranial Magnetic Stimulation

Transcranial Alternating Current Stimulation (tACS) is a neuromodulatory technique able to act through sinusoidal electrical waveforms in a specific frequency and in turn modulate ongoing cortical oscillatory activity. This neurotool allows the establishment of a causal link between endogenous oscillatory activity and behavior. Most of the tACS studies have shown online effects of tACS. However, little is known about the underlying action mechanisms of this technique because of the AC-induced artifacts on Electroencephalography (EEG) signals. Here we show a unique approach to investigate online physiological frequency-specific effects of tACS of the primary motor cortex (M1) by using single pulse Transcranial Magnetic Stimulation (TMS) to probe cortical excitability changes. In our setup, the TMS coil is placed over the tACS electrode while Motor Evoked Potentials (MEPs) are collected to test the effects of the ongoing M1-tACS. So far, this approach has mainly been used to study the visual and motor systems. However, the current tACS-TMS setup can pave the way for future investigations of cognitive functions. Therefore, we provide a step-by-step manual and video guidelines for the procedure.

Transcranial Alternating Current Stimulation (tACS) allows the modulation of cortical excitability in a frequency-specific fashion. Here we show a unique approach which combines online tACS with single pulse Transcranial Magnetic Stimulation (TMS) in order to "probe" cortical excitability by means of Motor Evoked Potentials.

Transcranial Alternating Current Stimulation (tACS) is a neuromodulatory technique able to act through sinusoidal electrical waveforms in a specific ...