This work has been funded by the Israel Science Foundation Grant (ISF # 393/12), Israel Centers of Research Excellence Grant (I-CORE 1796/12), German-Israel Foundation Grant (GIF # 2299-2291.1/2011) and the Marie Curie Career Integration Grant (FP7-PEOPLE-2013-CIG #618201). Initial steps in the project were funded by an AXA postdoctoral fellowship to AC. We acknowledge the generous startup funds provided by the Edmond and Lily Safra Center for Brain Sciences.
Critical reading by members of the Citri lab is greatly appreciated.

NOTE: The protocol follows the animal care guidelines of The Hebrew University of Jerusalem.
1. Preparation of ACSF Solution
<ol>
	<li>Prepare ACSF solution as described in Table 1. Make 1 L in ddH2O (&#62;18 M&#937; purity), bringing osmolarity to ~300 mOsm/L with appropriate addition of water or NaCl.</li>
</ol>
2. Equipment and Room Set Up
The equipment for monitoring of cocaine-induced locomotor behavior consis...

<ol>
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We have nothing to disclose.

Successful characterization of gene expression from brain tissue following behavioral paradigms depends upon: 1) Careful handling of mice during the behavioral paradigm; 2) Quick and precise dissection of tissue of interest; 3) RNA-safe measures to ensure the integrity of RNA; and 4) Careful planning of primers and experimental layout as well as precision and attention to detail in preparation for the qPCR analysis.
The aim of the procedure described is to characterize dynamics of transcriptio...

The brain&#8217;s dynamic organization enables cognitive and behavioral flexibility. Experiences are encoded through modifications of the structure and strength of connections between neurons in the brain3. This &#8220;experience-dependent plasticity&#8221; is the result of induction of specific patterns of gene expression that provides the necessary proteins for modification of synaptic structure and strength4. The identification of gene regulatory networks mediating the formation of long-term memories is a central tenet of molecular neuroscience, with the expectation that identification of the dominant elements of transcriptional programs will provide insight into the fundamental principles regulating memory formation, as well as targets for treatment of neurodegenerative and neuropsychiatric disorders. Transcriptional programs unfold in temporally-defined waves, each of which encode genes of different character, which are important for different stages in the implementation of the outcome of the signalling event1,2. It is therefore important to address transcriptional dynamics on a detailed temporal timescale, so as to identify the full complement of genes induced, and gain insight into their potential function according to the dynamics of their induction.
Drug addiction is a robust form of experience-dependent plasticity caused by the long-lasting effects of drugs of abuse on neural circuits in the brain5,6. Initial, acute exposure to drugs may lead to the development of addiction and the transition to chronic use. Contextual information is a crucial element in the development of addiction. Drug-associated environmental cues are assigned significant importance in the minds of drug abusers. Contextual information reminding a drug abuser of past drug experience can induce relapse to drug craving even following long periods of abstinence from drug exposure7,8. Hence the great clinical challenge in addiction - the propensity of addicts to relapse even long after withdrawal symptoms have subsided9.
Behavioral sensitization to cocaine is a simple model of cocaine experience useful in the study of mechanisms of drug addiction. In this widely-studied model for the long-lasting sensitization induced by chronic exposure to drugs of abuse, rodents are first habituated to saline injections (intraperitoneal; IP) in a novel environment (an open field chamber in which their locomotor activity is monitored); then, they receive daily injections of cocaine in the open field chambers while their activity is monitored10 (Figure 1). This behavioral paradigm typically results in a robust sensitization of locomotor behavior (8-12 fold above baseline activity)11, which is maintained for a period of months following cessation of cocaine injections, demonstrating the formation of a pervasive memory trace of drug experience.
The neural circuitry of reward, naturally involved in reinforcing behaviors essential for the success of a species (e.g. feeding, sex), is exploited by drugs of abuse to reinforce drug-associated behaviors12,13. The molecular and cellular mechanisms by which the experience of drugs of abuse is enhanced appear to be similar to the mechanisms underlying the formation of declarative or semantic memories in other brain structures14. Therefore, the robustness of the behavioral sensitization model makes it an attractive model system to study mechanisms of experience-dependent plasticity.
The nucleus accumbens (NAc) is a central integrator of the brain&#8217;s reward circuitry, and has been extensively associated with the development of addiction5,6. The formation of addiction depends on transcription of novel proteins in the nucleus accumbens, and robust induction of clearly structured transcription programs is observed in the NAc following cocaine experience15-19. The acute transcriptional response to cocaine exposure is likely to function at multiple levels in order to adapt to the strong induction stimulus and to direct the production of new proteins that are responsible for the structural and electrophysiological changes induced by exposure to the drug 6,19-22. 
In order to promote the study of molecular mechanisms of experience-dependent plasticity in the brain, a protocol is described for the comprehensive analysis of transcription dynamics in brain tissue samples following behavioral manipulation. The protocol is illustrated in the context of the behavioral experience studied in the Citri lab &#8211; behavioral sensitization to cocaine, utilizing microfluidic dynamic arrays for transcriptional analysis. The protocol described is obviously not limited to studying the nucleus accumbens in the context of behavioral sensitization, but could be applied to a large number of behavioral paradigms and brain regions. In fact, this protocol could be applied to body tissues outside the brain, and a variety of experiences or manipulations of the organism studied.
The protocol is roughly divided into four steps. In the first step, the animal is subjected to the behavioral paradigm; in the second step the tissue is microdissected; in the third step &#8211; mRNA is purified, reverse-transcribed and probed, and in the final step the data is analyzed.
In the context of studying transcriptional dynamics, the precise timing and definition of the experience are probably the most important experimental parameters to control. For this reason, our behavioral model of choice is that of behavioral sensitization to cocaine, a system which enables high level of experimenter control over the parameters of the experience. Additional behavioral paradigms that enable precise timing and address different models of experience-dependent plasticity or memory formation are available. These models include fear conditioning23, acute environmental enrichment24,25, novel object exploration26 and visual experience following dark rearing27. Still, behavioral sensitization to cocaine is a consistently robust behavioral manipulation, creating a highly pervasive memory trace which lasts for months following cocaine experience28.
The brain is sectioned, followed by manual microdissection of the nucleus accumbens. It has been our experience that manual microdissection from rapidly prepared brain slices provides the most reliable and rapid method of extracting the tissue relevant to the behavioral paradigm, and with experience, the boundaries of the tissue become evident and readily recognized. Alternatively, fine slices could be prepared, followed by laser-capture microdissection. Although this method enables highly defined delineation of the region of interest, it is slow (thus risking loss of labile mRNA), tedious and requires costly dedicated equipment (a microscope fitted with a laser-capture setup). The protocol defined herein could also be adapted to single-cell transcriptional analysis, by manual aspiration of cytoplasm of visually identified cells using patch pipettes29. It is important to note that the protocol described provides a population average, while it is highly likely that in most cases, only a subpopulation of the cells within the tissue are actually involved in responding to the experience. It is of interest to profile transcription in a selective manner from within specific cell populations responding to the experience, but discussion of these approaches is beyond the current scope.
For mRNA purification, reverse-transcription and qPCR querying, the tissue is disrupted by passing it through fine needles, followed by the utilization of commercially available kits (for more information, see Table 8). The choice is informed by experience with these methodologies, which ensure reliable extraction of high quality RNA and robust results from downstream applications.
While the protocol is described for high-throughput qPCR utilizing dynamic arrays, samples can be probed for gene expression using end-point PCR, low-throughput qPCR, gene expression microarrays or deep sequencing. The preference for high-throughput qPCR utilizing dynamic arrays is due to the fact that mRNA obtained from brain nuclei following behavioral paradigms is often of limiting quantities. Dynamic arrays provide a platform that enables efficient comprehensive analysis of transcripts from a large number of parallel samples in a single experiment. After the initial acquisition of the microfluidic system (commonly an institutional purchase), experiments are relatively inexpensive to run. Following this analysis, further querying of the samples could be performed using more costly platforms to search for novel transcripts (by microarrays or RNAseq) with the dynamic arrays providing a comprehensive reference for quality assurance. Finally, for data analysis, standard approaches are utilized. Specific pointers regarding issues that may arise will be discussed in the text of the protocol.
This protocol is most appropriate for investigators interested in a thorough investigation of their system of interest, studying multiple conditions and replicates. The protocol is also most suitable for investigators who have already honed in (through microarray or RNAseq experiments) on a subset of 50-500 genes of interest, which they are interested in querying repeatedly.

<table border="0" cellpadding="0" cellspacing="0" width="453">
	<tbody>
		<tr>
			<td>Virusol</td>
			<td>Oriek Medical</td>
			<td>J29D</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane, USP 100%</td>
			<td>MINRAD INC</td>
			<td>NDC 60307-110-25</td>
			<td></td>
		</tr>
		<tr>
			<td>RNeasy plus Universal Mini Kit</td>
			<td>QIAGENE</td>
			<td>73404</td>
			<td></td>
		</tr>
		<tr>
			<td>QIAshredder</td>
			<td>QIAGENE</td>
			<td>79654</td>
			<td></td>
		</tr>
		<tr>
			<td>High Capacity cDNA Reverse Transcription kit</td>
			<td>Invitrogene</td>
			<td>AB-4368814</td>
			<td></td>
		</tr>
		<tr>
			<td>TE Buffer</td>
			<td>Invitrogene</td>
			<td>1355656</td>
			<td></td>
		</tr>
		<tr>
			<td>Behaviour Chamber (MDF; 50X45cm)</td>
			<td>Self assembled</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Inner Perspex box (30X30cm)</td>
			<td>Self assembled</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>camera and video recorder</td>
			<td>Campden Inst</td>
			<td>CMD-80051</td>
			<td></td>
		</tr>
		<tr>
			<td>Media Recorder software</td>
			<td>Noldus</td>
			<td>NDS-NMR3-00M</td>
			<td></td>
		</tr>
		<tr>
			<td>Iris Scissors</td>
			<td>FST</td>
			<td>FST-14062-09</td>
			<td></td>
		</tr>
		<tr>
			<td>Sagital Brain slicer with a 0.5mm section</td>
			<td>Brain Tree Scientific</td>
			<td>BS-AL-505S</td>
			<td></td>
		</tr>
		<tr>
			<td>Bioanalyzer</td>
			<td>Agilent Technologies</td>
			<td>The Agilent 2100 Bioanalyzer</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermal cycler</td>
			<td>Bio-Rad</td>
			<td>1852048</td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted microspun spatula</td>
			<td>Bochem Instrument GmbH</td>
			<td>3213</td>
			<td></td>
		</tr>
		<tr>
			<td>Biomark HD Reader</td>
			<td>Fluidigm</td>
			<td>BMHD-BMKHD</td>
			<td></td>
		</tr>
		<tr>
			<td>Dynamic array Chip for 96.96gene expression</td>
			<td>Fluidigm</td>
			<td>BMK-M-96.96</td>
			<td></td>
		</tr>
	</tbody>
</table>

comprehensive analysis of transcription dynamics from brain samples following behavioral experience

The encoding of experiences in the brain and the consolidation of long-term memories depend on gene transcription. Identifying the function of specific genes in encoding experience is one of the main objectives of molecular neuroscience. Furthermore, the functional association of defined genes with specific behaviors has implications for understanding the basis of neuropsychiatric disorders. Induction of robust transcription programs has been observed in the brains of mice following various behavioral manipulations. While some genetic elements are utilized recurrently following different behavioral manipulations and in different brain nuclei, transcriptional programs are overall unique to the inducing stimuli and the structure in which they are studied1,2.
In this publication, a protocol is described for robust and comprehensive transcriptional profiling from brain nuclei of mice in response to behavioral manipulation. The protocol is demonstrated in the context of analysis of gene expression dynamics in the nucleus accumbens following acute cocaine experience. Subsequent to a defined in vivo experience, the target neural tissue is dissected; followed by RNA purification, reverse transcription and utilization of microfluidic arrays for comprehensive qPCR analysis of multiple target genes. This protocol is geared towards comprehensive analysis (addressing 50-500 genes) of limiting quantities of starting material, such as small brain samples or even single cells.
The protocol is most advantageous for parallel analysis of multiple samples (e.g. single cells, dynamic analysis following pharmaceutical, viral or behavioral perturbations). However, the protocol could also serve for the characterization and quality assurance of samples prior to whole-genome studies by microarrays or RNAseq, as well as validation of data obtained from whole-genome studies.

The quality of the results obtained by applying this protocol crucially depends upon a number of parameters. Proper experimental planning will result in minimal disturbance to the experimental mice, such that the experience tested (in this example, that of exposure to cocaine) will be the most dominant experience in their recent history, and therefore will result in a robust and specific transcriptional program. Figure 1 describes the experimental plan for behavioral sensitization to cocaine, defining th...

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Comprehensive Analysis of Transcription Dynamics from Brain Samples Following Behavioral Experience

This manuscript describes a protocol that applies comprehensive profiling for analysis of transcriptional programs induced in specific brain nuclei of rodents following behavioral paradigms. Herein, this approach is illustrated in the context of profiling genes induced in the nucleus accumbens (NAc) of mice following acute cocaine exposure, utilizing microfluidic qPCR arrays.

The encoding of experiences in the brain and the consolidation of long-term memories depend on gene transcription. Identifying the function of specific ...

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11.5K Görüntüleme.  The Hebrew University of Jerusalem. This manuscript describes a protocol that applies comprehensive profiling for analysis of transcriptional programs induced in specific brain nuclei of rodents following behavioral paradigms. Herein, this approach is illustrated in the context of profiling genes induced in the nucleus accumbens (NAc) of mice following acute cocaine exposure, utilizing microfluidic qPCR arrays. 