The authors would like to thank Gali Breuer for her assistance in the video production.&#160;This research was supported by the Israel Ministry of Science, Technology &#38; Space (grant no. 313552), by the National Institute for Psychobiology in Israel (NIPI-208-16-17b) and by the Open University Foundation.

All methods described here have been approved by the Institutional Animal Care and Use Committee of the Academic College Tel-Aviv-Yaffo.
1. Animals
<ol>
	<li>Use pre-adolescent (i.e., 3 weeks old) Institute of Cancer Research (ICR) outbred male mice.</li>
	<li>Randomize mice to two equally sized stress group (UCMS vs. na&#239;ve). Use 15 mice per treatment group (e.g.: if there are 3 pharmacological treatment groups use 90 mice overall; 2 [UCMS vs. na&#239;ve] &#215; 3 [tre...

Insofar as MDD is a widespread highly debilitating disorder, only partially addressed by current therapeutic options, the scientific quest for better treatments is still a pressing issue. Along with innovations in psychological techniques, additional pharmacotherapies are required for the large portion of patients who do not respond to the existing drugs. Meticulous animal models for depression are the key element in this task. Such models facilitate screenings for innovative antidepressants and expand the understanding ...

Major depressive disorder (MDD) is a debilitating condition, that has been indicated as the 11th cause of global burden from disease1, with a lifetime prevalence of 11&#8211;16%2,3. MDD has been associated with severe impairments on patients&#39; social and occupational functioning, diminished quality of life, numerous mental and physical disorders and increased risk for mortality4,5,6,7. There are several efficacious pharmacotherapies and psychological interventions for MDD; however, more than the third of the patients do not achieve remission with the existing therapeutic options8,9,10,11. Therefore, better mapping of the pathophysiology of MDD and development of novel drugs are still of utmost importance. In order to address these tasks scientifically validated animal models needs to be utilized.
Unpredictable chronic mild stress (UCMS) is a renowned rodent paradigm used to induce depressive- and anxiety-like behaviors12,13,14,15. The main objective of UCMS is to generate behavioral deficits (such as anhedonia and behavioral despair12,15) in mice and rats, and promote screenings for potential therapeutic pharmacological agents. The procedure was first introduced by Katz16 and subsequently developed by Willner17,18, yielding vast behavioral and neurobiological outcomes reminiscing depressive symptomatology12. It was initially designed for rats and later accommodated to mice13,19. In the procedure, adolescent animals are chronically exposed to different unpredictable mild stressors. Subsequently, pharmacological agents are administered. Behavioral and biological indices are obtained upon treatment termination. One of the more prominent tests conducted following UCMS is the sucrose preference test (SPT). The SPT is based on rodents&#39; innate preference for the sweetened solution rather than water and is widely acknowledged as an essential translational model for assessing anhedonia12,18,20,21 (which is a core symptom in human depression22,23).
While entering the fourth decade since its introduction, UCMS has been applied on mice and rats in myriad studies. The majority of these studies employed UCMS as a method to induce depressive-like behaviors12,13,21,24. Studies have also employed the model to generate anxiogenic effects25,26,27,28,29. Sucrose and saccharin preferences are the main tests used to assess anhedonia following UCMS12,18,30,31,32,33. Other notable outcome measures that are highly incorporated in UCMS literature are: the tail suspension test (TST)28,34,35, the forced swim test (FST)28,34,36,37 (both measuring stress coping/behavioral despair), the open field test (OFT; measuring exploratory behavior, anxiety-like behavior and locomotor activity)25,28,38, the elevated plus maze (EPM; measuring anxiety-like behavior)25,39,40 and additional tests measuring depressive-like behaviors, anxiety-like behaviors, cognitive functioning and social behavior12. Chronic administration of tricyclic antidepressants (TCAs; imipramine35,41,42,43, desipramine18,44,45), tetracyclic antidepressants (TeCAs; maprotiline46,47, mianserin48), selective serotonin reuptake inhibitors (SSRIs; fluoxetine46,47,49, escitalopram30,50, paroxetine51,52), melatonin43,49, agomelatine53, the fatty acid amide hydrolase (FAAH) inhibitor URB59754 and several natural compounds30,37,50,55,56,57,58 have been demonstrated to reverse the UCMS-induced depressive- and anxiety-like symptoms. Overall, these therapeutic effects have not been obtained via acute treatments12 (e.g., paroxetine51,52, imipramine53,54,59,60, fluoxetine53, agomelatine53, URB59754, brofaromine60).
Stress exposure during childhood and adolescence is a major risk factor for the anterior formation of MDD (among several other psychiatric disorders) in adulthood61,62,63. The hypothalamic-pituitary-adrenal (HPA) axis is a major neuroendocrine system regulating the bio-behavioral response to stress64. Long-term stress during the sensitive neurodevelopmental periods of childhood and adolescence impairs the equilibrium of the HPA axis. It might provoke a state of enhanced sympathetic activation, unbalanced reactivity and hypercortisolemia lasting through the resting state; thus, rendering individuals vulnerable to the depression or anxiety-related psychopathologies65,66,67,68. UCMS adequately translates this pathogenesis: stress application during mice&#39; adolescence induces a long-term depressive-like susceptibility. Moreover, the behavioral deficits induced by UCMS, are underlain by significant alterations in HPA axis functioning (e.g., by causing a reduction in hippocampal brain-derived neurotrophic factor [BDNF; a protein highly involved in the equilibrium of the HPA axis69,70]30, or by impairing the regulation of corticosterone secretion to the blood71,72), in similarity to the pathophysiology in humans12,50,73.
UCMS has several bolstering features as a model for depression: e.g. (i) the elicitation of anhedonia (which is regarded an endophenotype of MDD23,74); (ii) UCMS enables to assess wide variety of depressive-like behaviors such as behavioral despair, reduced social behavior, deterioration in fur state and more34; and (iii) chronic (2-4 weeks), but not acute, administration of antidepressants following stress exposure could produce a protracted therapeutic effect parallel to the effect obtained in human patients by the same agents30,75,76,77.
These features strengthen the validity of UCMS compared to other animal models of depression. The FST78 and the TST79 are two models that are used either to induce or to assess depressive-like behavior. As models for inducing depressive-like behaviors they have clear shortfalls compared to UCMS; they do not prompt long-term behavioral changes and might merely reflect an adjustment to acute stress rather than yield a durable depressive-like manifestation76.
An alternative animal model of depression is the social defeat model. Unlike the FST and the TST this model (like UCMS) require the application of chronic stress (id est [i.e.], the recurrent subjection of the animal to aversive social encounters with dominant counterparts)76,77,80,81,82. The main advantage of the social defeat model is that it employs social stimuli as stressors, thus reflecting the role of psychosocial stress in the pathogenesis of human depression. Similar to UCMS, the social defeat model elicits long-term depressive-like behaviors and neuroendocrine alterations. Yet again parallel to UCMS, the social defeat-induced deficits could be reversed via chronic, but not acute, administration of antidepressants. Overall, there is large support for the utilization of both UCMS and social defeat as pre-clinical apparatuses for investigating the pathophysiology of depression76,77,81,82. However, a major shortfall of the social defeat model is that it could only be applied on male rodents, as females do not exhibit sufficient aggressive behavior toward each other83. Contrastingly, UCMS has been shown to produce several depressive-like effects on both male and female mice34.
Predictable chronic mild stress (PCMS) is another rodent model that enforces a regimen of daily recurring exposure to restraint stress28,84,85,86,87. Several studies have shown that PCMS increased anxiety-like behaviors28,87; albeit, there are contradictory reports vis-&#224;-vis PCMS ability to induce long-term depressive-like behaviors. Unlike UCMS, PCMS has produced less satisfactory results referring to its ability to induce an anhedonic-like state28,84,86. This is consistent with the human phenomenology, in which unpredictable stressors are more harmful than predictable ones88.

<table>
	<tbody>
		<tr>
			<td>Heating lamp</td>
			<td>Ikea</td>
			<td>AA-19025-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Heating pillow</td>
			<td>Sachs</td>
			<td>EF-188B</td>
			<td></td>
		</tr>
		<tr>
			<td>Mice restrainer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Portable electronic balance (*.** g)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Standard rubber stopper, size 5</td>
			<td>Ancare</td>
			<td>#5.5R</td>
			<td>To avoid spillage during SPT</td>
		</tr>
		<tr>
			<td>Straight open drinking tube (2.5&#34;)</td>
			<td>Ancare</td>
			<td>OT-100</td>
			<td>To avoid spillage during SPT (insert drinking tube into rubber stopper)</td>
		</tr>
		<tr>
			<td>2% sucrose solution</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>50ml conical centrifuge tube</td>
			<td></td>
			<td></td>
			<td>For the SPT</td>
		</tr>
		<tr>
			<td>Pre-adolescent (approximately 20-days old) ICR outbred mice</td>
			<td>Envigo</td>
			<td>Hsd:ICR (CD-1)</td>
			<td></td>
		</tr>
	</tbody>
</table>

the unpredictable chronic mild stress protocol for inducing anhedonia in mice

Depression is a highly prevalent and debilitating condition, only partially addressed by current pharmacotherapies. The lack of response to treatment by many patients prompts the need to develop new therapeutic alternatives and to better understand the etiology of the disorder. Pre-clinical models with translational merits are rudimentary for this task. Here we present a protocol for the unpredictable chronic mild stress (UCMS) method in mice. In this protocol, adolescent mice are chronically exposed to interchanging unpredictable mild stressors. Resembling the pathogenesis of depression in humans, stress exposure during the sensitive period of mice adolescence instigates a depressive-like phenotype evident in adulthood. UCMS can be used for screenings of antidepressants on the variety of depressive-like behaviors and neuromolecular indices. Among the more prominent tests to assess depressive-like behavior in rodents is the sucrose preference test (SPT), which reflects anhedonia (core symptom of depression). The SPT will also be presented in this protocol. The ability of UCMS to induce anhedonia, instigate long-term behavioral deficits and enable reversal of these deficits via chronic (but not acute) treatment with antidepressants strengthens the protocol&#39;s validity compared to other animal protocols for inducing depressive-like behaviors.

In order to corroborate the efficacy of the UCMS procedure for inducing depressive-like deficits, a manipulation check was conducted. Male ICR outbred mice were randomly assigned to either UCMS or na&#239;ve conditions (4 weeks, as described in protocol&#160;2.2). Subsequently, the SPT (6 days, as described in protocol&#160;4) was administered to assess whether mice after undergoing UCMS demonstrated hedonic deficits. Shortly after, mice were sacrificed and the hippocampus was dissected o...

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The Unpredictable Chronic Mild Stress Protocol for Inducing Anhedonia in Mice

Here we present the unpredictable chronic mild stress protocol in mice. This protocol induces a long-term depressive-like phenotype and enables to assess the efficacy of putative antidepressants in reversing the behavioral and neuromolecular depressive-like deficits.

Depression is a highly prevalent and debilitating condition, only partially addressed by current pharmacotherapies. The lack of response to treatment by ...