MSB is supported by the Alcohol Beverage Medical Research Foundation, a Center for Translational Research Award (UL1 TR000058), the National Institutes for Alcohol Abuse and Alcoholism (P50 AA022537), and startup funds provided by the Virginia Higher Education Equipment Trust Fund and the VCU School of Medicine.

<ol>
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None of the authors have competing or conflicting interests.

The procedure presented here is an efficient means to manufacture microinjection cannulae and microinjectors that will aid in elucidating the molecular substrates of motivated behavior. This method offers several advantages. First, by manufacturing one&#8217;s own implants and microinjectors, novel experimental parameters can be rapidly optimized; i.e., one does not need to wait for custom made components to arrive. Second, due to the small diameter of the cannula, more cannulae can be simultaneously implanted. This shortens the required surgical time, which can improve survivability, and also allows multiple implants per animal. Third, the software used to control the operant chambers readily accommodates progressive ratio schedules since a fixed ratio paradigm can be rapidly converted to a progressive ratio paradigm by simply applying an event transition parameter list that contains the desired reinforcement schedule.
To be broadly useful, a generic microinjection procedure was presented that should be broadly applicable for the microinjection of nearly any reagent currently available. Consequently, we anticipate that this technique will continue to be of similar high utility in the future with minor modification. By changing only a few variables, this approach can be applied to a wide number of reagents. Parameters that would most commonly be manipulated include the length that the microinjector protrudes from the cannula, volume of injection, and rate of injection. For example, one may want the injector to protrude further from the cannula tip to avoid the glial scar that typically forms around chronic implants. Additionally, one may wish to inject a larger volume. For striatal virus microinjections, a volume of 1 &#956;L is typically used and this volume is typically injected over a longer period of time (frequently 7 &#8211; 10 min plus 3 &#8211; 10 min additional diffusion time) compared to that used for pharmacological reagents (typically 0.3 &#8211; 0.5 &#956;L over 2 &#8211; 3 min plus 1 &#8211; 3 min additional diffusion time). The user should consult the literature and/ or empirically determine the parameters best suited for their needs. Regardless, the success of this procedure is critically dependent upon 4 variables: 1) cannula length, 2) microinjector length, 3) quality of microinjector spray pattern, and 4) the system integrity prior to injection. Because microinjection location is dependent on the depth that the microinjector protrudes from the cannula, it is imperative that both cannulae (Step 1.2.8) and microinjector length (post-bending, Step 2.2.1) are both precisely known and uniform between all subjects. This can easily be controlled by readily rejecting any implement that is not the required length at the final re-measuring. Moreover, the injection location can only be predicted if it occurs immediately beneath the guide cannula. Thus, any microinjector that does not spray a long, fine stream upon testing (Steps 2.4.6) should be rejected. A quality injection is also related to the integrity of the system prior to injection. If after dispensing all water from the injector (prior to filling with reagent) multiple spots are observed on the lab-wipe, then a leak needs to be remedied (Note on Step 2.4.8). Further, if the bubble (Step 2.4.9) that separates the drug from the water in the PE20 tubing is not one, single bubble (after filling the microinjector with reagent), then the injector is partially clogged. This clog could either prevent or divert the injection. This too can be readily remedied (Note on step 2.4.8).
Should one wish to microinject while the animal is in the stereotaxic frame there are three alternatives. First, one could increase the length of the microinjector collar such that it can be held firmly by the stereotaxic manipulator and also extend far enough to allow connection to PE20 tubing. Second, one could temporally implant a cannula and use the standard microinjector presented here. Third, one could use drawn and polished glass pipettes. 16,17
A significant limitation of the procedure presented here is that it is best conducted in well-handled rats that are familiar with the procedure. Rats used for the data described in the results section required no special handling procedures because the same investigator handled the rats on a daily basis for over 2 months. This included daily observation and manipulation of the surgical implant for at least 2 weeks. However, rats can be rapidly habituated by a number of techniques that are used to prior to the pre-pulse inhibition assay, which can be affected by stress. These special habituation techniques have been nicely detailed previously.43 In addition to these procedures, it is advisable that rats be habituated to the microinjection procedure where shortened microinjectors are used during &#8216;sham&#8217; injections. During these sham injections, it is critical that the microinjector not protrude into the tissue in order to limit tissue damage. In other words, the microinjector should be bent no longer than 14 mm. Thus, the thorough habituation required for optimal implementation of this technique could be viewed as a limitation.
While several behavioral paradigms exist to measure motivation, the progressive ratio is commonly used to quantify the effort that the subject is willing to exert to obtain a reinforcer. The progressive ratio paradigm produces a measure known as breakpoint, which is often defined as the maximal number of lever presses in the last completed ratio; i.e., maximal responding that generated a reinforcer.21 The progressive ratio is sensitive to reinforcer magnitude. For example, higher cocaine (or sucrose) doses produce a higher breakpoint and lower cocaine (or sucrose) doses yield a lower breakpoint.21,22 Accordingly, breakpoint is a routinely used proxy for motivation and/or reinforcing efficacy.21,23-26 Because the intention of the breakpoint is to determine when the animal stops responding, an important parameter of the progressive ratio paradigm is session length. Finite session lengths can put a false cap on breakpoint values and this can be exacerbated by pre-treatments that abnormally decrease the rate of self-administration or that increase post-reinforcement pausing. This confound can be overcome by any number of approaches; e.g., sessions that terminate when the animal has withheld responding by some multiple of the average inter-infusion interval.44 A more commonly applied variant of this approach is to terminate sessions once responding has been withheld by some empirically determined value that is held constant across subjects. We have provided the method to apply this approach in Step 4.4.9.11.

Rodents and humans respond in remarkably similar ways to behaviorally relevant stimuli1-3. This suggests that rodents are appropriate subjects for elucidating the molecular substrates of behavior and complex psychiatric conditions4. Understanding the molecular substrates of complex behavioral processes, such as motivation, frequently requires brain microinjection. Both the brain microinjection technique and a primary motivation assay will be presented here. Rats will be used as subjects, but these procedures can readily be adapted to well-handled mice. Included herein are procedures for the manufacture of the required cannulae, obturators (dummy cannulae or stylets), and microinjectors. The method presented is significantly more flexible and more cost-efficient than prefabricated implements. This flexibility will prove valuable when optimizing conditions. Importantly, because the microinjection procedure can be used to test a myriad of hypotheses; the techniques presented here should be broadly applicable. For example, receptor ligands can be microinjected to understand neurochemistry3,5,6; cell-permeable peptides and small-molecules can be microinjected to understand intracellular signaling pathways7-10; toxins, ion channel blockers, or antagonist cocktails can be microinjected to understand circuitry1,11,12.
While the generic protocol presented here can be readily adapted by the user for their particular needs, the procedure is particularly well suited for behavioral assays since microinjection occurs in awake rodents that are only under mild hand restraint. No anesthesia or special restraints are required. This is possible because the brain itself lacks pain sensation. However, if anesthesia is not used, microinjection must occur through cannulae that were previously stereotaxically implanted. This is because nociceptors are present on the scalp, meninges,13 which are the membranes surrounding the brain, and the periosteum,14 which is the membrane covering the skull. It should be noted that microinjection under anesthesia is sometimes desirable. One example is when the virus is being injected, and one may wish to inject virus directly through either stainless steal needles15 or glass pipettes because this can reduce tissue damage and improve transduction efficiency.16,17 The microinjectors described below can be modified for this purpose and suggestions on how to do this can be found in the Discussion. Because other JoVE articles have demonstrated stereotaxic brain cannula implantation,18-20 these procedures will not be covered here.
We present these microinjection procedures together with an assay that quantifies motivation. Several rodent models of motivated behavior are currently in use, such as the runway box and barrier scaling. Here, we describe how to use an operant progressive ratio schedule of reinforcement to quantify motivation where operant responding is being maintained by a reinforcer. Responding on the progressive ratio is responsive to reinforcer magnitude.21,22 Accordingly, this assay is routinely used as a proxy for motivation and/or reinforcing efficacy. 21,23-30 Because several excellent reviews have covered this topic in detail,21,24 we will focus mainly on practical concerns.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Cannula Tubing</td>
			<td>Amazon Supply/ Small Parts</td>
			<td>HTXX-26T-60</td>
			<td>26 gauge, Hypotube S/S 316-TW 26GA</td>
		</tr>
		<tr>
			<td>Obturator</td>
			<td>Amazon Supply/ Small Parts</td>
			<td>GWXX-0080-30-05</td>
			<td>33 gauge, Wire S/S 316LVM 0.008 IN</td>
		</tr>
		<tr>
			<td>Microinjector Wire</td>
			<td>MicroGroup</td>
			<td>33RW 304</td>
			<td>33 gauge</td>
		</tr>
		<tr>
			<td>Super Glue</td>
			<td>Loctite</td>
			<td>3924AC</td>
			<td>Liquid, Non-gel, can be autoclaved</td>
		</tr>
		<tr>
			<td>Microinjector Plastic Tubing</td>
			<td>Becton Dickson</td>
			<td>427406</td>
			<td>PE20</td>
		</tr>
		<tr>
			<td>Medium Weight Hemostats</td>
			<td>World Precision Instruments</td>
			<td>501241-G</td>
			<td></td>
		</tr>
		<tr>
			<td>Ruler</td>
			<td>Fisher</td>
			<td>09-016</td>
			<td>150 mm</td>
		</tr>
		<tr>
			<td>#7 Forceps</td>
			<td>Stoelting</td>
			<td>52100-77</td>
			<td>Dumont, Dumostar</td>
		</tr>
		<tr>
			<td>Rotary Tool</td>
			<td>Dremmel</td>
			<td>285</td>
			<td>Two-speeds</td>
		</tr>
		<tr>
			<td>Cut-off Disc</td>
			<td>McMaster Carr</td>
			<td>3602</td>
			<td>15/16&#34; x 0.025&#34;</td>
		</tr>
		<tr>
			<td>Microinjection Pump</td>
			<td>Harvard Apparatus</td>
			<td>PhD 2000</td>
			<td></td>
		</tr>
		<tr>
			<td>1 ul Glass Syringe</td>
			<td>Hamilton</td>
			<td>7001KH</td>
			<td>Needle Style: 25s/2.75&#34;/3</td>
		</tr>
		<tr>
			<td>Cotton Tipped Applicator</td>
			<td>Fisher</td>
			<td>23-400-101</td>
			<td></td>
		</tr>
		<tr>
			<td>Lab Wipes</td>
			<td>Kimwipes</td>
			<td>34133</td>
			<td></td>
		</tr>
		<tr>
			<td>Operant Software</td>
			<td>Coulbourn</td>
			<td>Graphic State</td>
			<td></td>
		</tr>
		<tr>
			<td>Operant Chambers</td>
			<td>Coulborun</td>
			<td>Habitest</td>
			<td></td>
		</tr>
	</tbody>
</table>

rodent brain microinjection to study molecular substrates of motivated behavior

Brain microinjection can aid elucidation of the molecular substrates of complex behaviors, such as motivation. For this purpose rodents can serve as appropriate models, partly because the response to behaviorally relevant stimuli and the circuitry parsing stimulus-action outcomes is astonishingly similar between humans and rodents. In studying molecular substrates of complex behaviors, the microinjection of reagents that modify, augment, or silence specific systems is an invaluable technique. However, it is crucial that the microinjection site is precisely targeted in order to aid interpretation of the results. We present a method for the manufacture of surgical implements and microinjection needles that enables accurate microinjection and unlimited customizability with minimal cost. Importantly, this technique can be successfully completed in awake rodents if conducted in conjunction with other JoVE articles that covered requisite surgical procedures. Additionally, there are many behavioral paradigms that are well suited for measuring motivation. The progressive ratio is a commonly used method that quantifies the efficacy of a reinforcer to maintain responding despite an (often exponentially) increasing work requirement. This assay is sensitive to reinforcer magnitude and pharmacological manipulations, which allows reinforcing efficacy and/ or motivation to be determined. We also present a straightforward approach to program operant software to accommodate a progressive ratio reinforcement schedule.

Here we illustrate examples of results that can be obtained with the above procedure. Shown first is a typical area that is transfected by viral vectors (Figure 3). In general, a transfected volume of ~1 mm3 is obtained in striatal tissue. The volume of the transfected region can be quantified across serial sections using Cavalieri&#8217;s method.32 Importantly, the transfected volume depends on many factors such as the type of tissue being transfected; viral serotype; promoter; rate and volume injected; number of particles injected; injectate pH; and whether hyperosmolar reagents, such as mannitol, were used.33,34 Typically, we microinject 1012 particles/ml, 1 &#181;l/side over 10 min, and allow 7 additional min prior to replacing obturators. Additionally, the injectate is generally around pH 8. Next, we show that motivation can be manipulated by either transgene expression (Figure 4) or pharmacological reagent microinjection (Figure 5).
In Figure 4, we expressed a Designer Receptor Exclusively Activated by Designer Drugs (DREADDs). The DREADD receptor-coding region was followed by an Internal Ribosome Entry Site and a mCitrine cassette. The mCitrine allows convenient visualization of transfected cells. The DREADD was coupled to the heterotrimeric G-protein G&#945;q. Activation of the G&#945;q-coupled DREADD can stimulate astrocytes,28,35 and the DREADD itself can be activated by systemic administration of clozapine-N-oxide (CNO, 3 mg/kg, i.p).14,22,36 Rats were trained to lever respond for ethanol reinforcement, where 3 lever presses yielded 1 chance to drink during daily 1 hr sessions over 60 contiguous days. Next, rats were forced into abstinence, and G&#945;q-coupled DREADDs were expressed in nucleus accumbens core astrocytes. After 3 week abstinence, the motivation to self-administer ethanol was measured by breakpoint.21,27-30 Activation of nucleus accumbens core astrocytes, via systemic CNO administration, decreased the motivation of rats to resume ethanol self-administration after abstinence compared to vehicle. Importantly, CNO had no effect in an equally trained cohort that was expressing Green Fluorescent Protein instead of the DREADD.
<img alt="Figure 3" src="/files/ftp_upload/53018/53018fig3.jpg" /> 
Figure 3. Representative ventral striatal region transfected by microinjected virus. This image illustrates the region of nucleus accumbens astrocytes that were transfected by following the above method. Data are reprinted from Bull et al.13 with permission of the copyright holder. Details can be found in that publication and the accompanying supplementary material. <a href="https://www.jove.com/files/ftp_upload/53018/53018fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/53018/53018fig4.jpg" /> 
Figure 4. Motivation of rats to self-administer ethanol was reduced by activating a transgene that was over-expressed in nucleus accumbens core astrocytes. Virus was microinjected and motivation measured via breakpoint after allowing one week for the virus to express. The transgene expressed was a Designer Receptor Exclusively Activated by Designer Drugs (DREADDs). A one-way ANOVA (F(2,30)=3.29, p=0.04) followed by a Scheff&#233; post-hoc revealed that activation of the DREADD by systemic administration of clozapine-N-oxide (CNO, 3 mg/kg, ip) significantly reduced the motivation of rats to self-administer ethanol after abstinence compared to vehicle CNO had no effect in an equally trained cohort that was expressing Green Fluorescent Protein (GFP) instead of the DREADD. Data are reprinted from Bull et al.13 with permission of the copyright holder. Details can be found in that publication and the accompanying supplementary material.
<img alt="Figure 5" src="/files/ftp_upload/53018/53018fig5.jpg" /> 
Figure 5. Microinjection of gap junction blockers increased the motivation of rats to self-administer ethanol after abstinence. Two gap junction blockers were evaluated for their effect on the motivation (measured via breakpoint) of rats to self-administer ethanol: mefloquine and 18-&#945;-glycyrrhetinic acid (18-&#945;). A two-way ANOVA revealed that microinjection of gap-junction blockers into the nucleus accumbens core increased the motivation of rats to self-administer ethanol after abstinence (F(3,40)=5.56, p=0.003). Data are reprinted from Bull et al.13 with permission of the copyright holder. Details can be found in that publication and the accompanying supplementary material.

Watch this Scientific Journal Video about Rodent Brain Microinjection to Study Molecular Substrates of Motivated Behavior at JoVE.com

Rodent Brain Microinjection to Study Molecular Substrates of Motivated Behavior

Rodents are an appropriate model to investigate the molecular substrates of behavior and complex psychiatric disorders. Brain microinjection in awake rodents can be used to elucidate disease substrates. An efficient and customizable brain microinjection method as well as the execution of an operant paradigm that quantifies motivation is presented.

Brain microinjection can aid elucidation of the molecular substrates of complex behaviors, such as motivation. For this purpose rodents can serve as ...

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JoVE Journal

Behavior

14.1K 조회수.  Virginia Commonwealth University School of Medicine. Rodents are an appropriate model to investigate the molecular substrates of behavior and complex psychiatric disorders. Brain microinjection in awake rodents can be used to elucidate disease substrates. An efficient and customizable brain microinjection method as well as the execution of an operant paradigm that quantifies motivation is presented. 