NIH R01MH084894 participated in the funding of this study. We would like to thank Drs. Yasmin Hurd and Scott Russo at Mount Sinai School of Medicine for the donation of mice and the use of their surgery and behavior facilities during the filming of this work.

NOTE: All procedures for animal breeding and cares were conducted according to the Institutional Animal Care and Use Committee&#160;(IACUC) regulation of Icahn School of Medicine at Mount Sinai. Be sure to use sterile gloves throughout the procedure.&#160;
1. Drug and Virus Preparation
<ol>
	<li>Drug Preparation
	<ol>
		<li>Prepare 15.0 ml ketamine/xylazine anesthetic by dissolving 1.35 ml of 100 mg/ml Ketamine and 0.75 ml of 20 mg/ml xylazine in 12.9 ml of 0.9% Saline solution. Thoroughly mix solution.</li>
	</ol>
	</li>
	<li>Virus Preparation
	<ol>
		<li>Clone the mGlu2 and mGlu2&#916;TM4N constructs into a bicistronic herpes simplex virus (HSV) vector following standard protocols previously described6. Package the viral particles as previously described6,13,14. Substitution of residues Ala-6774.40, Ala-6814.44 and Ala6854.48 in mGlu2 for Ser6864.40, Phe6904.44 and Gly-6944.48 in mGlu3 (HA-mGlu2&#916;TM4N) have been described previously6. 
		NOTE: It was previously demonstrated that the chimeric construct HA-mGlu2&#916;TM4N is expressed at the plasma membrane with intact G protein-dependent signaling6.</li>
		<li>Store viral vectors in -80 &#176;C when not in use. Thaw viral vector on ice, and then aliquot into 10 &#956;l aliquots. For the surgical procedure, keep on ice.</li>
	</ol>
	</li>
</ol>
2. Surgery
<ol>
	<li>Surgery Preparation
	<ol>
		<li>Weigh mouse and inject mouse with appropriate dose of ketamine/xylazine cocktail (for details, see 1.1.1).</li>
		<li>Check mouse to see if properly anesthetized, squeeze foot and tail for pain response, if unable to elicit a response, the mouse is properly anesthetized.</li>
		<li>Shave mouse head from the base of the skull to the tip of the nose using clippers. Apply ophthalmic gel to the mouse afterward to prevent blindness of the mouse.</li>
		<li>Load each syringe onto the stereotaxic frame. Then tilt perpendicular portion of each arm of the stereotaxic frame so that they are 10 degrees away from the normal. Ensure that the arms are tilted, such that the needles are facing each other.</li>
		<li>Clean each syringe by filling the needle with 70% ethanol. Fill the needle at least three times to ensure that the syringe is clean.</li>
		<li>Once the needle has been cleaned, flush the needle by filling the needle with double distilled H2O. Once flushed, fill each needle with 1.3 &#181;l of double distilled H2O. Twist the plunger of the syringe to release 0.3 &#181;l of double distilled H2O. If water beads at the tip of the needle, carefully wipe away water. If nothing comes out of the syringe, push the plunger completely down and then repeat cleaning of syringe.</li>
		<li>After filling with water, then pull up the syringe filling the syringe with 0.5 &#181;l of air.</li>
		<li>Once the air and water are in the syringe, carefully fill the syringe with 1.3 &#181;l of virus solution. At this point ensure that the total volume in the syringe is 2.8 &#181;l. Again twist the tip of the syringe to release 0.3 &#181;l of virus. If liquid beads at the tip of the needle, carefully wipe away liquid. If nothing comes out of the syringe, push the plunger completely down and then repeat cleaning of syringe.</li>
	</ol>
	</li>
	<li>Surgery
	<ol>
		<li>Attach the mouse to the stereotaxic frame, making sure to adjust the stereotaxic frame so that the skull is level and flat. Apply povodine-iodine to the exposed scalp. Using a scalpel, make a sagittal incision along the midline of the skull within the exposed shaved area. Then attach the buret clamps to the skin at the incision site to make sure that the skull remains exposed.</li>
		<li>Use H2O2 to dissolve away the periosteum to expose the sutures of the skull. Now that the bregma and sutures are visible, be sure to adjust the stereotaxic frame to make sure that the skull is level.</li>
		<li>Align the needle tips of the syringes with the bregma and record the coordinates of the bregma. Calculate the coordinates of the where the needles are going to be inserted.
		<ol>
			<li>For the Rostral-Cauldal (R-C) plane, add 1.6 mm to the recorded R-C bregma coordinates (+1.6 from Bregma). For the Dorsal-Ventral (D-V) plane, subtract 2.4 mm from the recorded D-V bregma coordinates (-2.4 from Bregma).</li>
			<li>Finally, for the Medial Lateral (M-L) plane, add 2.6 mm to the recorded M-L bregma coordinates (+2.6 from Bregma). For all coordinates be sure to record both left and right coordinates, as this is a bilateral injection.</li>
		</ol>
		</li>
		<li>Bring the needles to the desired coordinates. Mark the places of where the needles are going to be inserted and with a drill, drill the marked areas.</li>
		<li>With a cotton tip applicator wipe away any excess blood or bone fragment.</li>
		<li>Bring the needles to the skull where the tips of the needles are touching the surface of the brain. Then lower the needles to the desired coordinates slowly lowering them.</li>
		<li>Once the needles are at the desired coordinates, slowly inject the contents of the syringe by twisting the plunger of the needle 0.1 &#181;l per minute over the course of 5 min&#160;(in total 0.5 &#181;l).</li>
		<li>Once the injection has been made leave the syringe in cortex for another 5 min.</li>
	</ol>
	</li>
	<li>Closing Up/Care
	<ol>
		<li>Remove the needles from the mouse cortex steadily and slowly. Then remove the mouse from the stereotaxic frame.</li>
		<li>Apply cyanoacrylate (dermal adhesive) to the base flaps of skin from the incision and then with forceps grab the flaps of skin and place them together.</li>
		<li>Allow the cyanoacrylate to dry. Place the mouse in cage over a heating pad (heating pad is optional if the surgical suite is kept under RT of 37 &#176;C - otherwise not necessary). Be sure to place the mouse on a paper towel to make sure that bedding does not adhere to surgical site.</li>
		<li>Depending on the length of the surgical procedure, ensure that the mice is out of anesthesia within 30 - 60 min&#160;after procedure.&#160;Following surgery, the animal is placed in its own cage and monitored until it becomes conscious before returning to its room to recover. No analgesics are used post-operatively, because they may alter the results of our experiments by modifying some of the brain biochemical pathways. However, animals are monitored until they recover from anesthesia on the day of surgery, and daily post-operation for signs of infection and evaluation of pain/distress.</li>
	</ol>
	</li>
</ol>
3. Head Twitch Response Experiment
<ol>
	<li>Set-up
	<ol>
		<li>Carry out all behavioral testing between 10:00 AM and 2:00 PM, 2 - 3 days after stereotactic injection of viral particles.</li>
		<li>Dissolve (&#177;)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane hydrochloride (DOI) into a 0.9% saline solution to 2.0 mg/kg. Also prepare a 0.9% saline solution.</li>
		<li>Prepare a home cage (28 x 18 x 15 cm) without any bedding and using a tri-pod, adjust a camera so that the view of the video camera is directly above the home cage.</li>
		<li>Habituate the mice to the room for at least 4 hr prior to the beginning of the experiment.</li>
		<li>Set up a camcorder to record the head twitch.</li>
	</ol>
	</li>
	<li>Experiment
	<ol>
		<li>Position the camera so that it is directly over a home cage. Calibrate the camera so that the entire home cage is in the field of view.</li>
		<li>Weigh mouse and inject mouse intraperitoneally with appropriate dose of either 0.9% saline or DOI (0.01 ml/g). NOTE: If a mouse weighs 25 grams, administer dose to a total volume of 0.25 ml.</li>
		<li>Place each mouse back in their home cage for 10 min. After 10 min, place mouse into the center of the empty home cage and validate that there are no blind spots in the field of view of the camera. Press record on the camcorder. Leave the room. 
		NOTE: Mouse movements and various behavioral responses therein (head twitch, ear scratch, etc. Please refer to supplemental data table 1 of Gonzalez-Maeso et al. 2007 for full list of behavioral responses induced by DOI.)3, will be recorded for 30 min. Therefore, it is important there are no blind spots in the field of view recorded.</li>
		<li>After 30 min&#160;stop recording on the camcorder and place mouse back in original home cage. Repeat this process for each mouse.</li>
	</ol>
	</li>
	<li>Review
	<ol>
		<li>Have each referee review the tapes blind to the experimental conditions of mouse (i.e., drugs used during head twitch experiment or virus used during intracranial injection)). Manually record every head twitch throughout the video. 
		NOTE: Head-twitch is defined as a rapid shaking head movement conducted by a mouse (supplemental video).</li>
		<li>For each mouse, average the final HTR response from the three totals of the blind referees. Then group these values by experimental condition and carry out statistical analysis (i.e., t-test or ANOVA).</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

Together with previous findings in mGlu2-KO mice5, the results with mGlu2 and mGlu2/mGlu3 chimeric constructs that do not form the 5-HT2A-mGlu2 receptor complex in cultured cells suggest that the 5-HT2A -mGlu2 heteromeric receptor complex in mouse frontal cortex is needed to induce head-twitch behavior by LSD-like hallucinogenic 5-HT2A receptor agonists. A limitation of this method is that it does not measure close molecular proximity at a subcellular level in native tissue. In addition, there are various critical points to be noted. Because the mice are injected with a HSV viral vector, the time frame that the experiments to be performed are 2 - 4 days after injection. The location and expression of the viral vectors should be verified with immunofluorescence staining of sectioned brain no more than 4 days after the initial injection. Mice in which the coordinates do not match or do not express the viral vector should be excluded from the experimental data as they do not express the mGlu2 or mGlu2&#916;TM4N. Care after stereotactic surgery is also crucial, as improper closing of the head wound can lead to infection which can cause issues in both the in vivo experiments and the immunofluorescence staining. Lastly, after stereotactic injection any behavioral paradigm (open field, alternating t-test, etc.) can be used as long as it is within the 2 - 4 after injection of viral particles. Again, coordinates and expression should be confirmed by immunofluorescence.
The concept that GPCRs function as homo- and/or heteromers in living cells is now well established 16-18. However, despite some progress, more studies are needed to define the precise role(s) of GPCR heteromeric complexes in whole animal models 19. Approaches such as BRET and/or FRET imaging to investigate protein-protein physical proximity within deep tissues of small animal models may provide means to enhance the understanding of the functional role of GPCR heteromers in living subjects20. Using HSV-mediated expression of GPCRs either native or with modified transmembrane proteins, provide an in vivo model to evaluate the function and interaction of GPCRs.
Further studies in rodent models are also needed to examine the stability and life- time (formation and dissociation) of GPCR heteromers21-23, structural rearrangements between their components24, and potential G protein coupling after receptor internalization25,26. Given the fundamental role of GPCRs in cell signaling and function, it seems likely that this area might lead to interesting basic and translational studies.
Although further investigation is required to quantitatively characterize the ultrastructural co-localization of both receptors in human and mouse CNS, together with previous studies that convincingly demonstrate the electrophysiological, cellular, and behavioral responses induced by hallucinogens in mouse models are intrinsic to 5-HT2A receptor-expressing cortical pyramidal neurons3,27,28, the findings obtained using the HSV-mediated expression approach described here suggest that heteromeric formation between 5-HT2A and mGlu2 receptors in mouse frontal cortex is needed for the head-twitch psychosis-like behavior induced by the hallucinogenic 5-HT2A receptor agonist DOI.
HSV-mediated expression of mGlu2, but not mGlu2&#916;TM4N, in frontal cortical neurons of mGlu2-KO mice rescues the head-twitch behavior induced by the hallucinogenic 5-HT2A receptor agonist DOI. This translational tool might be advantageous for preclinical studies to evaluate behavioral phenotypes of GPCR heteromers.

Hallucinogens, such as LSD, psilocybin and mescaline cause significant changes in human consciousness, cognition and emotion7-9. Inactivation of serotonin 5-HT2A receptor signaling by either genetic or pharmacological approaches causes markedly attenuated behavioral responses to hallucinogens in both rodent models3,10 and humans11. Although hallucinogens bind other receptor subtypes8, the 5-HT2A receptor is considered as necessary for the unique behavioral activity of these chemicals.
Group II metabotropic glutamate receptors (i.e., mGlu2 and mGlu3) have been the target of considerable attention regarding the molecular mechanism of hallucinogens and their integral role underlying psychosis12. Previously, it has been demonstrated that mice with no expression of mGlu2 protein (mGlu2-KO mice) are insensitive to the cellular and behavioral effects of hallucinogens5. It has also been suggested that the 5-HT2A and the mGlu2 receptors form a specific heteromeric complex through which serotonin and glutamate ligands modulate the pattern of G protein coupling in living cells 1,2.
Structurally, transmembrane (TM) domains 4 and 5 of mGlu2 play a fundamental role in heteromeric formation with the 5-HT2A receptor5. Additionally, further investigation demonstrated that three residues located at the intracellular end of TM4 of mGlu2 are necessary to form the 5-HT2A-mGlu2 receptor heterocomplex in living cells6.
Based on these findings observed in heterologous expression systems, here we describe the use of HSV-mediated expression of wild-type mGlu2 and mGlu2/mGlu3 chimeric constructs in the frontal cortex of mGlu2-KO mice to test whether heteromeric formation between 5-HT2A and mGlu2 is necessary for the head-twitch behavior induced by hallucinogenic 5-HT2A receptor agonists.

<table border="0" cellpadding="0" cellspacing="0" width="2329">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">mGlu2 bicitronic herpes simplex virus (HSV) vector&#160;</td>
			<td style="width:71px;">MIT Core</td>
			<td style="width:119px;"></td>
			<td style="width:1716px;">mGlu2 and mGlu2DTM4N were subcloned into the bicistronic HSV-GFP virus vector p1005+ HSV expressing GFP under the control of the CMV promoter. Viral particles were produced by the Viral Core Facility at the McGovern Institute (MIT). For more information, please contact the director, Dr. Rachael Neve (rneve@mit.edu)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">mGlu2&#916;TM4N bicitronic herpes simplex virus (HSV) vector&#160;</td>
			<td style="width:71px;">MIT Core</td>
			<td style="width:119px;"></td>
			<td>mGlu2 and mGlu2DTM4N were subcloned into the bicistronic HSV-GFP virus vector p1005+ HSV expressing GFP under the control of the CMV promoter. Viral particles were produced by the Viral Core Facility at the McGovern Institute (MIT). For more information, please contact the director, Dr. Rachael Neve (rneve@mit.edu)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">GFP bicitronic herpes simplex virus (HSV) vector&#160;</td>
			<td style="width:71px;">MIT Core</td>
			<td style="width:119px;"></td>
			<td>mGlu2 and mGlu2DTM4N were subcloned into the bicistronic HSV-GFP virus vector p1005+ HSV expressing GFP under the control of the CMV promoter. Viral particles were produced by the Viral Core Facility at the McGovern Institute (MIT). For more information, please contact the director, Dr. Rachael Neve (rneve@mit.edu)</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:423px;">xylazine&#160;</td>
			<td style="width:71px;">Lloyd</td>
			<td style="width:119px;">List no. 4811-20ml, NADA #139-236, NDC Code(s): 61311-481-10</td>
			<td>1.35 mL of 100mg/ml of ketamine+.75 mL of 20mg/ml of xylazine are diluted in 12.0 mL of .9% saline solution</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;">ketamine&#160;</td>
			<td style="width:71px;">Vedco</td>
			<td style="width:119px;">KetaVed-10ml, NADA #200-029, NDC Code(s): 50989-161-06</td>
			<td>1.35 mL of 100mg/ml of ketamine+.75 mL of 20mg/ml of xylazine are diluted in 12.0 mL of .9% saline solution</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">ophthalmic gel</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td>NC0550805</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">burret clips</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td>NC9268369</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:423px;">Feather surgical blade</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td>NC9032736</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:423px;">Hydrogen Peroxide</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td>19-898-919&#160;</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Hamilton syringe</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td>14815203</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Hamilton&#8482; Small Hub Removable Needles (33 Ga)</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td>14816206</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Cordless Micro Drill</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td>NC9089241</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Dermabond Dermal Adhesive</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td style="width:119px;">NC0690470</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">(&#177;)-1-(2,5-Dimethoxy-4-iodophenyl)-2-aminopropane hydrochloride (DOI)</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td>42203-78-1</td>
			<td>Dissolved in .9% saline solution to the concentration of 2.0 mg/kg</td>
		</tr>
	</tbody>
</table>

hsv-mediated transgene expression of chimeric constructs to study behavioral function of gpcr heteromers in mice

The heteromeric receptor complex between 5-HT2A and mGlu2 has been implicated in some of the behavioral phenotypes in mouse models of psychosis1,2. Consequently, investigation of structural details of the interaction between 5-HT2A and mGlu2 affecting schizophrenia-related behaviors represents a powerful translational tool. As previously shown, the head-twitch response (HTR) in mice is elicited by hallucinogenic drugs and this behavioral response is absent in 5-HT2A knockout (KO) mice3,4. Additionally, by conditionally expressing the 5-HT2A receptor only in cortex, it was demonstrated that 5-HT2A receptor-dependent signaling pathways on cortical pyramidal neurons are sufficient to elicit head-twitch behavior in response to hallucinogenic drugs3. Finally, it has been shown that the head-twitch behavioral response induced by the hallucinogens DOI and lysergic acid diethylamide (LSD) is significantly decreased in mGlu2-KO mice5. These findings suggest that mGlu2 is at least in part necessary for the 5-HT2A receptor-dependent psychosis-like behavioral effects induced by LSD-like drugs. However, this does not provide evidence as to whether the 5-HT2A-mGlu2 receptor complex is necessary for this behavioral phenotype. To address this question, herpes simplex virus (HSV) constructs to express either mGlu2 or mGlu2&#916;TM4N (mGlu2/mGlu3 chimeric construct that does not form the 5-HT2A-mGlu2 receptor complex) in the frontal cortex of mGlu2-KO mice were used to examine whether this GPCR heteromeric complex is needed for the behavioral effects induced by LSD-like drugs6.

Previous findings demonstrate that the head-twitch murine behavioral response is reliably and robustly elicited by hallucinogens, and it is absent in 5-HT2A-KO mice3. Furthermore, it has been shown that the head-twitch response elicited by the hallucinogenic 5-HT2A agonists DOI and LSD was significantly decreased in mGlu2-KO mice5. However, although previous findings convincingly demonstrate that 5-HT2A and mGlu2 are assembled as a heteromeric complex in vitro in transfected cells1,2,15, whether this structural arrangement behaves as such in living mice remained unsolved. To fully understand the role of the 5-HT2A-mGlu2 receptor heterocomplex in the psychoactive-like effects induced by hallucinogenic 5-HT2A receptor agonists, expression of either mGlu2 or mGlu2&#916;TM4N in frontal cortex of mGlu2-KO mice to examine whether this manipulation regulates behavior.
Mice received intra-frontal cortical injections of bicistronic HSV expressing green fluorescent protein (GFP) and either mGlu2 or mGlu2&#916;TM4N, or GFP alone. First, it was confirmed that the virus over-expresses mGlu2 or mGlu2&#916;TM4N in mouse frontal cortex (Figures 1A and 1B). As previously demonstrated5, head-twitch behavior induced by DOI was absent in mGlu2-KO mice injected with the empty vector HSV-GFP. Notably, the head-twitch response induced by the hallucinogenic 5-HT2A agonist DOI was rescued in mGlu2-KO mice over-expressing mGlu2, but not mGlu2&#916;TM4N, in frontal cortex as compared to that seen in animals expressing GFP (Figure&#160;1C). Together, these findings suggest that the 5-HT2A-mGlu2 receptor complex in frontal cortex is critical for regulating psychosis-like states.
<img alt="Figure 1" src="/files/ftp_upload/53717/53717fig1.jpg" /> 
Figure 1. Expression of mGlu2 as a Receptor Heterocomplex. Expression of mGlu2 as a receptor with 5-HT2A is necessary for psychosis-like behavior induced by hallucinogenic drugs. (A) Representative image of HSV-mediated transgene expression in frontal cortex. HSV-mGlu2, which also expresses GFP, was injected into frontal cortex, and GFP expression was revealed by immunocytochemistry, scale bar, 200-um. (B) HSV-mediated transgene expression in mouse frontal cortex of mGlu2-KO mice, and anti-mGlu2 reactivity was measured by Western Blotting. Specificity of the primary antibody against the mGlu2 receptor has previously been confirmed in knockout mice6. Metabotropic glutamate receptors are GPCRs that form covalently linked homodimers. We measured immunoreactivity of mGlu2 as a monomer (100 kDa)6. (C) Viral-mediated expression of mGlu2, but not mGlu2&#916;TM4N, in frontal cortex of mGlu2- KO mice significantly rescues the head-twitch response induced by the hallucinogenic 5-HT2A agonist DOI (n = 4 per group). ***p &#60;0.001; n.s, not significant; Bonferroni&#39;s post hoc test of one-way ANOVA. Error bars represent S.E.M. Figure was modified from Moreno et al (2012)6. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53717/53717fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53717/HeadTwitchResponse.wmv" target="_blank">Supplemental Video 1. Head Twitch Response. (Right click to download).</a> CD-1 WT mice were injected with 2.0 mg/kg DOI and placed in a cage (wall blacked out between the two cages) to elicit head-twitch response (behavior elicited after *).

Watch this Scientific Journal Video about HSV-Mediated Transgene Expression of Chimeric Constructs to Study Behavioral Function of GPCR Heteromers in Mice at JoVE.com

HSV-Mediated Transgene Expression of Chimeric Constructs to Study Behavioral Function of GPCR Heteromers in Mice

This article describes how to inject viral vectors into the mouse frontal cortex to test behavioral assays that require GPCR heteromeric formation.

The heteromeric receptor complex between 5-HT2A and mGlu2 has been implicated in some of the behavioral phenotypes in mouse models of psychosis1,2.  ...

hsv-mediated-transgene-expression-chimeric-constructs-to-study

Nanyang Technological University

Research

JoVE Journal

Neuroscience

7.2K Views.  Icahn School of Medicine at Mount Sinai. This article describes how to inject viral vectors into the mouse frontal cortex to test behavioral assays that require GPCR heteromeric formation.

Video: HSV-Mediated Transgene Expression of Chimeric Constructs to Study Behavioral Function of GPCR Heteromers in Mice

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 ...

Local Application of Drugs to Study Nicotinic Acetylcholine Receptor Function in Mouse Brain Slices

Tobacco use leads to numerous health problems, including cancer, heart disease, emphysema, and stroke. Addiction to cigarette smoking is a prevalent neuropsychiatric disorder that stems from the biophysical and cellular actions of nicotine on nicotinic acetylcholine receptors (nAChRs) throughout the central nervous system. Understanding the various nAChR subtypes that exist in brain areas relevant to nicotine addiction is a major priority.
Experiments that employ electrophysiology techniques such as whole-cell patch clamp or two-electrode voltage clamp recordings are useful for pharmacological characterization of nAChRs of interest. Cells expressing nAChRs, such as mammalian tissue culture cells or Xenopus laevis oocytes, are physically isolated and are therefore easily studied using the tools of modern pharmacology. Much progress has been made using these techniques, particularly when the target receptor was already known and ectopic expression was easily achieved. Often, however, it is necessary to study nAChRs in their native environment: in neurons within brain slices acutely harvested from laboratory mice or rats. For example, mice expressing "hypersensitive" nAChR subunits such as &alpha;4 L9&prime;A mice 1 and &alpha;6 L9&prime;S mice 2, allow for unambiguous identification of neurons based on their functional expression of a specific nAChR subunit. Although whole-cell patch clamp recordings from neurons in brain slices is routinely done by the skilled electrophysiologist, it is challenging to locally apply drugs such as acetylcholine or nicotine to the recorded cell within a brain slice. Dilution of drugs into the superfusate (bath application) is not rapidly reversible, and U-tube systems are not easily adapted to work with brain slices.
In this paper, we describe a method for rapidly applying nAChR-activating drugs to neurons recorded in adult mouse brain slices. Standard whole-cell recordings are made from neurons in slices, and a second micropipette filled with a drug of interest is maneuvered into position near the recorded cell. An injection of pressurized air or inert nitrogen into the drug-filled pipette causes a small amount of drug solution to be ejected from the pipette onto the recorded cell. Using this method, nAChR-mediated currents are able to be resolved with millisecond accuracy. Drug application times can easily be varied, and the drug-filled pipette can be retracted and replaced with a new pipette, allowing for concentration-response curves to be created for a single neuron. Although described in the context of nAChR neurobiology, this technique should be useful for studying many types of ligand-gated ion channels or receptors in neurons from brain slices.

In this paper, we describe a useful method to study ligand-gated ion channel function in neurons of acutely isolated brain slices. This method involves the use of a drug-filled micropipette for local application of drugs to neurons recorded using standard patch clamp techniques.

Tobacco use leads to numerous health problems, including cancer, heart disease, emphysema, and stroke. Addiction to cigarette smoking is a prevalent ...

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, ...

A Simple Behavioral Assay for Testing Visual Function in Xenopus laevis

Measurement of the visual function in the tadpoles of the frog, Xenopus laevis, allows screening for blindness in live animals. The optokinetic response is a vision-based, reflexive behavior that has been observed in all vertebrates tested. Tadpole eyes are small so the tail flip response was used as alternative measure, which requires a trained technician to record the subtle response. We developed an alternative behavior assay based on the fact that tadpoles prefer to swim on the white side of a tank when placed in a tank with both black and white sides. The assay presented here is an inexpensive, simple alternative that creates a response that is easily measured. The setup consists of a tripod, webcam and nested testing tanks, readily available in most Xenopus laboratories. This article includes a movie showing the behavior of tadpoles, before and after severing the optic nerve. In order to test the function of one eye, we also include representative results of a tadpole in which each eye underwent retinal axotomy on consecutive days. Future studies could develop an automated version of this assay for testing the vision of many tadpoles at once.

A Simple Behavioral Assay for Testing Visual Function in Xenopus laevis

Xenopus laevis tadpoles prefer swimming on the white side of a black/white tank. This behavior is guided by their vision. Based on this behavior, we present a simple assay to test the visual function of tadpoles.

Measurement of the visual function in the tadpoles of the frog, Xenopus laevis, allows screening for blindness in live animals. The optokinetic response ...

Assessment of Dendritic Arborization in the Dentate Gyrus of the Hippocampal Region in Mice

Dendritic arborization has been shown to be a reliable marker for examination of structural and functional integrity of neurons. Indeed, the complexity and extent of dendritic arborization correlates well with the synaptic plasticity in these cells. A reliable method for assessment of dendritic arborization is needed to characterize the deleterious effects of neurological disorders on these structures and to determine the effects of therapeutic interventions. However, quantification of these structures has proven to be a formidable task given their complex and dynamic nature. Fortunately, sophisticated imaging techniques can be paired with conventional staining methods to assess the state of dendritic arborization, providing a more reliable and expeditious means of assessment. Below is an example of how these imaging techniques were paired with staining methods to characterize the dendritic arborization in wild type mice. These complementary imaging methods can be used to qualitatively and quantitatively assess dendritic arborization that span a rather wide area within the hippocampal region.

We describe two methods for visualization and quantification of dendritic arborization in the hippocampus of mouse models: real-time and extended depth of field imaging. While the former method allows sophisticated topographical tracing and quantification of the extent of branching, the latter allows speedy visualization of the dendritic tree.

Dendritic arborization has been shown to be a reliable marker for examination of structural and functional integrity of neurons. Indeed, the complexity ...

Quantification of Filamentous Actin (F-actin) Puncta in Rat Cortical Neurons

Filamentous actin protein (F-actin) plays a major role in spinogenesis, synaptic plasticity, and synaptic stability. Changes in dendritic F-actin rich structures suggest alterations in synaptic integrity and connectivity. Here we provide a detailed protocol for culturing primary rat cortical neurons, Phalloidin staining for F-actin puncta, and subsequent quantification techniques. First, the frontal cortex of E18 rat embryos are dissociated into low-density cell culture, then the neurons grown in vitro for at least 12-14 days. Following experimental treatment, the cortical neurons are stained with AlexaFluor 488 Phalloidin (to label the dendritic F-actin puncta) and microtubule-associated protein 2 (MAP2; to validate the neuronal cells and dendritic integrity). Finally, specialized software is used to analyze and quantify randomly selected neuronal dendrites. F-actin rich structures are identified on second order dendritic branches (length range 25-75 &#181;m) with continuous MAP2 immunofluorescence. The protocol presented here will be a useful method for investigating changes in dendritic synapse structures subsequent to experimental treatments.

Quantification of Filamentous Actin F-actin Puncta in Rat Cortical Neurons

Filamentous actin (F-actin) plays an important role in spinogenesis, synaptic plasticity, and synaptic stability. Quantification of F-actin puncta is therefore a useful tool to study the integrity of synaptic structures. This protocol describes the procedures of quantifying F-actin puncta labeled with Phalloidin in low-density primary cortical neuronal cultures.

Filamentous actin protein (F-actin) plays a major role in spinogenesis, synaptic plasticity, and synaptic stability. Changes in dendritic F-actin rich ...

A Free-breathing fMRI Method to Study Human Olfactory Function

The study of human olfaction is a highly complex and valuable field with applications ranging from biomedical research to clinical evaluation. Currently, evaluation of the functions of the human central olfactory system with functional magnetic resonance imaging (fMRI) is still a challenge because of several technical difficulties. There are some significant variables to take into account when considering an effective method for mapping the function of the central olfactory system using fMRI, including proper odorant selection, the interaction between odor presentation and respiration, and potential anticipation of or habituation to odorants. An event-related, respiration-triggered olfactory fMRI technique can accurately administer odorants to stimulate the olfactory system while minimizing potential interference. It can effectively capture the precise onsets of fMRI signals in the primary olfactory cortex using our data post-processing method. The technique presented here provides an efficient and practical means for generating reliable olfactory fMRI results. Such a technique can ultimately be applied in the clinical realm as a diagnostic tool for diseases associated with olfactory degeneration, including Alzheimer&#39;s and Parkinson&#39;s disease, as we begin to further understand the complexities of the human olfactory system.

We present the technical challenges and solutions for obtaining reliable functional magnetic resonance imaging (fMRI) data from the human central olfactory system. This includes special considerations in olfactory fMRI paradigm design, descriptions of fMRI data acquisition with an MRI-compatible olfactometer, odorant selection, and a special software tool for data post-processing.

The study of human olfaction is a highly complex and valuable field with applications ranging from biomedical research to clinical evaluation. Currently, ...

Adeno-associated Virus-mediated Transgene Expression in Genetically Defined Neurons of the Spinal Cord

Selective manipulation of spinal neuronal subpopulations has mainly been achieved by two different methods: 1) Intersectional genetics, whereby double or triple transgenic mice are generated in order to achieve selective expression of a reporter or effector gene (e.g., from the Rosa26 locus) in the desired spinal population. 2) Intraspinal injection of Cre-dependent recombinant adeno-associated virus (rAAV); here Cre-dependent AAV vectors coding for the reporter or effector gene of choice are injected into the spinal cord of mice expressing Cre recombinase in the desired neuronal subpopulation. This protocol describes how to generate Cre-dependent rAAV vectors and how to transduce neurons in the dorsal horn of the lumbar spinal cord segments L3-L5 with rAAVs. As the lumbar spinal segments L3-L5 are innervated by those peripheral sensory neurons that transmit sensory information from the hindlimbs, spontaneous behavior and responses to sensory tests applied to the hindlimb ipsilateral to the injection side can be analyzed in order to interrogate the function of the manipulated neurons in sensory processing. We provide examples of how this technique can be used to analyze genetically defined subsets of spinal neurons. The main advantages of virus-mediated transgene expression in Cre transgenic mice compared to classical reporter mouse-induced transgene expression are the following: 1) Different Cre-dependent rAAVs encoding various reporter or effector proteins can be injected into a single Cre transgenic line, thus overcoming the need to create several multiple transgenic mouse lines. 2) Intraspinal injection limits manipulation of Cre-expressing cells to the injection site and to the time after injection. The main disadvantages are: 1) Reporter gene expression from rAAVs is more variable. 2) Surgery is required to transduce the spinal neurons of interest. Which of the two methods is more appropriate depends on the neuron population and research question to be addressed.

Intraspinal injection of recombinase dependent recombinant adeno-associated virus (rAAV) can be used to manipulate any genetically labelled cell type in the spinal cord. Here we describe how to transduce neurons in the dorsal horn of the lumbar spinal cord. This technique enables functional interrogation of the manipulated neuron subtype.

Selective manipulation of spinal neuronal subpopulations has mainly been achieved by two different methods: 1) Intersectional genetics, whereby double or ...

The Power of Interstimulus Interval for the Assessment of Temporal Processing in Rodents

Temporal processing deficits have been implicated as a potential elemental dimension of higher-level cognitive processes, commonly observed in neurocognitive disorders. Despite the popularization of prepulse inhibition (PPI) in recent years, many current protocols promote using a percent of control measure, thereby precluding the assessment of temporal processing. The present study used cross-modal PPI and gap prepulse inhibition (gap-PPI) to demonstrate the benefits of employing a range of interstimulus intervals (ISIs) to delineate effects of sensory modality, psychostimulant exposure, and age. Assessment of sensory modality, psychostimulant exposure, and age reveals the utility of an approach varying the interstimulus interval (ISI) to establish the shape of the ISI function, including increases (sharper curve inflections) or decreases (flattening of the response amplitude curve) in startle amplitude. Additionally, shifts in peak response inhibition, suggestive of a differential sensitivity to the manipulation of ISI, are often revealed. Thus, the systematic manipulation of ISI affords a critical opportunity to evaluate temporal processing, which may reveal the underlying neural mechanisms involved in neurocognitive disorders.

Temporal processing, a preattentive process, may underlie deficits in higher-level cognitive processes, including attention, commonly observed in neurocognitive disorders. Using prepulse inhibition as an exemplar paradigm, we present a protocol for manipulating interstimulus interval (ISI) to establish the shape of the ISI function to provide an assessment of temporal processing.

Temporal processing deficits have been implicated as a potential elemental dimension of higher-level cognitive processes, commonly observed in ...

Ballistic Labeling of Pyramidal Neurons in Brain Slices and in Primary Cell Culture

It has been reported that the size and shape of dendritic spines is related to their structural plasticity. To identify the morphological structure of pyramidal neurons and dendritic spines, a ballistic labeling technique can be utilized. In the present protocol, pyramidal neurons are labeled with DilC18(3) dye and analyzed using neuronal reconstruction software to assess neuronal morphology and dendritic spines. To investigate neuronal structure, dendritic branching analysis and Sholl analysis are performed, allowing researchers to draw inferences about dendritic branching complexity and neuronal arbor complexity, respectively. The evaluation of dendritic spines is conducted using an automatic assisted classification algorithm integral to the reconstruction software, which classifies spines into four categories (i.e., thin, mushroom, stubby, filopodia). Furthermore, an additional three parameters (i.e., length, head diameter, and volume) are also chosen to assess alterations in dendritic spine morphology. To validate the potential of wide application of the ballistic labeling technique, pyramidal neurons from in vitro cell culture were successfully labeled. Overall, the ballistic labeling method is unique and useful for visualizing neurons in different brain regions in rats, which in combination with sophisticated reconstruction software, allows researchers to elucidate the possible mechanisms underlying neurocognitive dysfunction.

We present a protocol to label and analyze pyramidal neurons, which is critical for evaluating potential morphological alterations in neurons and dendritic spines that may underlie neurochemical and behavioral abnormalities.

It has been reported that the size and shape of dendritic spines is related to their structural plasticity. To identify the morphological structure of ...