anxiety-related interventions in rodent defense behaviors: systematic review and meta ... · this...

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Anxiety-related interventions in rodent defense behaviors:

systematic review and meta-analyses

Authors

Farhan Mohammad1, Joses Ho2, Chun Lei Lim, Jia Hern Woo, Dennis Jun Jie Poon2, Bhumika Lamba2 & Adam Claridge-Chang1, 2, 3, 4

Affiliations

1. Program in Neuroscience and Behavioral Disorders, Duke-NUS Graduate Medical School, Singapore 138673 2. Institute for Molecular and Cell Biology, Agency for Science Technology and Research, Singapore 138673 3. Department of Physiology, National University of Singapore, Singapore 138673 4. Corresponding author: [email protected]

Keywords anxiety, defense, behavior, rodent, stress, serotonin, meta-analysis

.CC-BY 4.0 International licensewas not certified by peer review) is the author/funder. It is made available under aThe copyright holder for this preprint (whichthis version posted November 18, 2015. . https://doi.org/10.1101/020701doi: bioRxiv preprint

https://doi.org/10.1101/020701

http://creativecommons.org/licenses/by/4.0/

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ABSTRACT

Background

Assays measuring defense behavior in rodents, including the elevated plus maze, open field and light-dark box assays, have been widely used in preclinical models of anxiety to study the ability of therapeutic interventions to modulate the anxiety-like state. However, many important proposed anxiety-modulating factors, including genes, drugs and stressors have had paradoxical effects in these assays across different studies. Objectives

We performed a systematic review and meta-analysis of the literature on the effect of anxiety-targeted interventions on rodent defense behaviors in three commonly-used assays: the elevated plus maze, open field and light-dark box assays. Our aim was to determine the effect sizes of a panel of purported anxiety-related interventions across three assays. Data Sources

Using PubMed and EMBASE search phrases to identify articles, a systematic review was conducted on a panel of ten anxiety-linked interventions: diazepam, 5-HT1A receptor knockout and overexpression, SERT knockout and overexpression, pain, restraint, social isolation, corticotropin-releasing hormone and Crhr1. In addition, an ad hoc literature search was used to identify articles studying the effects of two genes that are not related to anxiety behaviours in rodents: adenylyl cyclase 1 and synaptotagmin.

Study Eligibility Criteria

Articles were included if they contained data on the effects of any one of the above-mentioned anxiety factors on one of the three rodent defense behaviors. Synthesis Methods

Systematic meta-analyses were conducted; synthesis of the data was performed using random effects models of Hedges’ g. Results

Eight of the ten anxiety-related interventions had statistically significant effects on rodent defense behavior, while Htr1a overexpression and Crh knockout did not. Evidence for publication bias was found in three interventions: diazepam, Htt knockout, and social

isolation. Limitations


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This meta-analysis excluded unpublished and non-English studies; publication bias and heterogeneity were identified in several meta-analyses. Conclusions

The synthetic data support eight of the ten proposed anxiety factors as having moderate or large effects on rodent anxiety as measured by ARDEB assay (diazepam, Htr1a knockout, Htr1a overexpression, Htt knockout, Htt overexpression, pain, restraint and Crhr1

knockout). Two interventions has small effects on rodent anxiety: social isolation and Crh knockout.


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INTRODUCTION

The anxiety disorders are among the costliest classes of mental disorders, both in morbidity and economic cost (DiLuca and Olesen 2014; Baldwin et al. 2014). Development of anxiety-reducing (anxiolytic) drugs has been a major focus of the pharmaceutical industry and academic neuropsychiatric research, though no new drug types have been adopted since the introduction of selective serotonin uptake inhibitors (SSRIs) and other antidepressants for the treatment of anxiety disorders (Tone 2009; Griebel and Holmes 2013). Anxiety research relies on similarities between human emotional behavior and behaviors in animals

(Darwin 1998), specifically rat and mouse (Prut and Belzung 2003). While there are many rodent behavioral paradigms that aim to model anxiety-like behaviors, three related assays that specifically aim to measure anxiety-related defense behavior (ARDEB) have been established, also referred to as the approach-avoidance conflict tests (Griebel and Holmes 2013). These assays are the elevated plus maze (EPM), the light-dark box (LD) and the open field (OF), the first, second and fifth most widely used assays in animal anxiety research, respectively (Griebel and Holmes 2013). All three arena types used in these assays contain a sheltered domain (e.g., the closed arms in EPM) and an exposed region. It is thought that avoidance of the exposed portions of the chamber reports on anxiety-like states in the rodent brain. These assays are generally accepted as preclinical assays of anxiety disorders, by reference to classic studies that validated them with a panel of drugs known to have anxiety-modulating effects in humans (Crawley and Goodwin 1980; Pellow et al.

1985; Simon, Dupuis, and Costentin 1994). Rodent research has been implicated in the largely frustrated efforts to develop new types of anxiolytics (Griebel and Holmes 2013). The literature regarding defense behavior is contradictory about the size and even the direction of many interventions that are proposed to be anxiolytic or anxiogenic (together ‘anxiotropic’) (Prut and Belzung 2003; Griebel and Holmes 2013). This is true even for some anxiety-related genes with major clinical relevance, such as Htt, the target of SSRIs. As with the assessment of clinical anxiety interventions (Baldwin et al. 2014), a solid evidence base of the effect of ‘mainstream’ anxiety interventions in the rodent ARDEB assays is necessary to guide decisions about further basic research and therapeutic development (Vesterinen et al. 2014). In light of the

widespread discordance, we aimed to conduct a quantitative review of some factors purported to influence rodent anxiety as measured by ARDEB. The primary aim of this study was to examine the validity of purported anxiety-influencing factors and to estimate


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the magnitude of their effects on rodent anxiety. A secondary goal of this analysis were to examine patterns in ARDEB factor evidence: gaps in the literature, the extent of standardization/heterogeneity and publication bias. Establishing the validity of anxiety-targeted interventions might assist in understanding why these assays have not led to new therapies. Once identified by meta-analysis, effective anxiotropic interventions can be also adopted as benchmarks against which to validate new rodent assays and/or more genetically tractable model animal species (e.g. Drosophila and zebrafish).


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METHODS

Literature review

Genes, drugs and environmental interventions that had been proposed to be involved in anxiety were identified by a literature search of review articles on the field of anxiety. Based on the history of anxiety research (Tone 2009; Griebel and Holmes 2013), a list of ten anxiotropic interventions were selected to be included in the systematic review, either due to their direct clinical importance (e.g., diazepam), their role as an example of a class of proposed anxiety-related factors (e.g., isolation), or their connection to a clinically important drug (e.g., Htt). A systematic review was conducted to identify published articles addressing experimental outcomes in rodents from the EPM, OF, or LD assays for these interventions. A comprehensive literature set for each genetic, pharmacological or environmental intervention was generated by a search on PubMed and EMBASE using specific search phrases (Table 1). The selective serotonin reuptake inhibitors (SSRIs) will be the subject of a separate study currently in preparation, due to their clinical importance

(Baldwin et al. 2014), the very large number of studies conducted on them (Griebel and

Holmes 2013), and their controversial efficacy (Kirsch et al. 2008).

Eligibility criteria and study selection

Bibliographic data (including study ID, date of publication, title and abstract) of studies identified in the systematic review were exported to a spreadsheet using the database export function. Each article on this list was then reviewed at four levels of detail (title, abstract, full text and a detailed review of experimental design) to determine their eligibility for the review. Studies were required to be written in English and to have reported ARDEB in adult rats or mice. We required that each included study contain (1) primary behavior data from either an OF, EPM, or LD experiment for the intervention of interest, (2) suitable controls were reported and (3) the relevant statistics were reported (mean, standard error or standard deviation, and sample sizes of both control and intervention groups). Since the focus of this study was the direct effects of anxiety-related interventions on ARDEB, experiments that used combination treatments were excluded. For drug and environmental interventions, possible confounding effects of the role of developmental stage were reduced by excluding studies not performed in adult rodents. For gene knockout and overexpression interventions, only experiments using a lifetime loss of function were included in the meta-analyses. We include all eligible studies in the meta-analysis (Table 1).


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Selection of non-anxiety-related interventions

Studies of the effect of non-anxiety-related interventions on ARDEB were harder to find, perhaps due to the bias against publishing negative results, and/or reduced likelihood of mentioning them in abstracts. PubMed and EMBASE searches in this area failed to identify any useful studies. Instead, a conventional ad hoc literature search was used to find reports of the effects of adenylyl cyclase 1 (two articles) and synaptotagmin (one article) on anxiety-related behaviors.

Data items and extraction

The following data were collected from each of the included studies: authors, year of publication, figure and panel numbers, species, genotype, and mean, standard error of the mean and sample size (N) of each intervention and its related control group. Graphically presented data were extracted from Portable Document Format (PDF) files with the Measuring Tool in Adobe Acrobat Pro. All extracted data were checked by a second researcher. For values extracted from tables, the check consisted of ensuring the values were identical. For values extracted from graphical data (e.g. bar plots), the check consisted of a visual inspection to ensure that the value extracted with matched that of the graphical data. Any data discrepancy was reconciled by discussion and inspection by the original data extractor and the researcher who identified the discrepancy.

Summary measures

The following behavioral metrics were extracted from the articles: in OF studies, percent or total time spent at the center; in EPM studies, percent or total time spent on the open arm; in LD studies, percent or total time spent in the bright area. To synthesize these time-based metrics from the three assays, all estimates were standardized to Hedges’ g, where g is a preferred variant of Cohen’s d that uses the pooled standard deviation and is corrected for bias using Hedges’ method (Cumming 2012; Borenstein et al. 2011). The conventional

adjectives to describe effect size (trivial, small, moderate, large) are used where appropriate (Cumming 2012).

Synthesis of results

Meta-analyses of experimental outcomes, including the calculation of weighted mean effect sizes (Hedges’ g), 95% confidence intervals, I2 heterogeneity values, and p values using the random effects model, were performed with the metafor package in R (http://CRAN.R-project.org/package=metafor) (Viechtbauer 2010). All error bars in forest plots are 95%


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confidence intervals; forest plots were generated with metafor and custom R scripts, and formatted in Adobe Illustrator.

Assessment of bias across studies

Publication bias was assessed using funnel plots and Egger’s linear regression test for funnel plot asymmetry (Egger et al. 1997). The standard normal deviate (Hedges’ g / standard error) for each study was regressed against the study’s precision (1 / standard error) using the “lm” function in R (http://www.R-project.org/). For studies that had evidence

of publication bias (p-value ≤ 0.05), the trim-and-fill method (Duval and Tweedie 2000) was employed to estimate the effects of publication bias on the effect size estimate. Funnel plots and trim-and-fill adjustments were performed with the ‘metafor’ package in R (Viechtbauer 2010).


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RESULTS

Review selection criteria identified 298 eligible articles

The flow-chart in Figure 1 summarizes the study selection process. In total, 1155 articles were identified by the initial search in PubMed and EMBASE databases. According to the selection criteria described above, 500 studies were excluded based on their titles and a further 149 were excluded based on their abstracts. The full text of the remaining 506 articles were screened for criteria related to experimental paradigm, methods, and relevant variables, resulting in the exclusion of a further 224 studies. A total of 282 articles were considered eligible for inclusion in the review.

Characteristics of included experiments

The characteristics of all included studies are given in Table 3. In brief, 297 studies comprising 412 EPM experiments, 85 OF experiments and 86 LD experiments were identified. Studies were published between 1985 and 2015 and included data from 293 experiments conducted on mice and 287 experiments on rats. Studies reported 516 experiments conducted on male animals, 29 on female, 34 on mixed and 3 experiments with no gender information reported. ARDEB studies of diazepam used a median dosage of 1 mg/kg, with minimum and maximum dosages of 0.01 mg/kg and 20 mg/kg respectively. This dose range is similar to or higher than commonly used by patients.

Heterogeneity

Statistically significant heterogeneity was found in (8/10) of the meta-analyses. Only two meta-analyses had high heterogeneity (I2 > 75%): Htr1a overexpression, and physical

restraint (Higgins et al. 2003). Three of the meta-analyses, pain and Htt knockouts and diazepam, had moderate heterogeneity (50% < I2 < 75%). Five meta-analyses had low heterogeneity (I2 < 50%). As most of these syntheses contained data from more than one assay type, it is encouraging that half had low or moderate heterogeneity; this outcome is compatible with the idea that the three ARDEB assays are testing similar aspects of rodent anxiety.

Publication bias

Censorship of statistically non-significant experimental results and selective publication of statistically significant ‘positive’ results can cause a literature (and meta-analyses thereof)


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to overstate effect sizes. For example, this effect (publication bias) has a profound influence on the literature on rodent models of stroke (Sena et al. 2010). Publication bias in the ARDEB literature was assessed for the six meta-analyses that had at least ten experiments (Table 2) (Sterne et al. 2011). Funnel plots of these data also showed pronounced

asymmetry (Figure 2), a property that can arise from publication bias, the censorship of experiments showing small and/or statistically non-significant effect sizes (the file drawer effect) (Sterne et al. 2011). Egger’s linear regression test for asymmetry revealed that three of these literatures showed statistically significant bias (Table 2). For the three biased data sets, we applied trim-and-fill adjustment to correct for the funnel plot asymmetry and to estimate the number of hypothesized missing studies (Duval and Tweedie 2000). We conclude that the literatures of diazepam, Htt knockout and social isolation effects on ARDEB are affected by publication bias.

Diazepam produces a moderate reduction in defense behaviors

Diazepam is an important minor tranquilizer that was used for several decades as the first line of treatment for anxiety disorders (Tone 2009) and, along with other benzodiazepines, is still used extensively to control anxiety (Baldwin et al. 2014). Recent clinical meta-analysis

studies have found support for the efficacy of benzodiazepines in the short-term treatment of anxiety disorders (Baldwin et al. 2014). However, a published review of diazepam effects in open field studies revealed widespread disagreement between with 29 studies supporting an anxiolytic effect and 23 supporting either an anxiogenic effect or no effect

(Prut and Belzung 2003). We reviewed the available literature on diazepam for the three major rodent ARDEB assays: EPM, OF and LD. This review identified 172 articles containing relevant data (de A Vieira et al. 2013; de Almeida et al. 2012; Assie et al. 1993;

Bahi et al. 2014; Barbosa et al. 2008; Baretta et al. 2012; Barnes et al. 1990; Bellavite et al. 2011; Belzung and Agmo 1997; Bhatt et al. 2013; Bhattacharya and Mitra 1991; Birkett et al. 2011; Blainski et al. 2010; Borsini et al. 1993; Brioni et al. 1994; Carneiro et al. 2005;

Carro-Juarez et al. 2012; de Castro et al. 2007; Cechin et al. 2003; Cha et al. 2005; Chen et al. 2005; Chen et al. 2004; Choleris et al. 2001; Cole and Rodgers 1995; Colla et al. 2015; Consoli et al. 2007; Contreras et al. 2011; Costa et al. 2011; Costall et al. 1990; Dalvi and

Rodgers 2001; Dalvi and Rodgers 1999; de-Paris et al. 2000; Drapier et al. 2007; R. W. Dunn et al. 1998; R. W. Dunn, Corbett, and Fielding 1989; Ene et al. 2015; Engin, Treit, and Dickson 2009; Ennaceur et al. 2010; Fajemiroye et al. 2014; Faria et al. 1997; Faturi et al.

2010; F. Fernandez et al. 2004; S. P. Fernandez et al. 2008; Flores et al. 2006; Fortes et al.


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2013; Fraser et al. 2010; Frassetto et al. 2010; Galeotti, Sanna, and Ghelardini 2013; Girish et al. 2013; Gomes et al. 2010; González-Pardo, Conejo, and Arias 2006; Ma Eva Gonzalez-

Trujano et al. 2006; Maria Eva Gonzalez-Trujano et al. 2015; Griebel, Perrault, Tan, Schoemaker, and Sanger 1999b; Griebel, Perrault, Tan, Schoemaker, and Sanger 1999a; Griebel, Perrault, and Sanger 1998; Griebel, Perrault, and Sanger 1997; Griebel et al. 2002; Guilloux et al. 2013; Gupta et al. 2015; Gupta, Radhakrishnan, and Kurhe 2014; Han et al.

2009; Harada et al. 2006; Hasenohrl et al. 1996; Hazim et al. 2014; Huerta-Reyes et al. 2013; Hui et al. 2002; Ishaq 2014; N. S. Jain, Hirani, and Chopde 2005; Jastrzebska-Wiesek et al. 2014; Jászberényi et al. 2009; Jászberényi et al. 2007; Jessa et al. 1996; Jones et al.

1994; Kalouda and Pitsikas 2015; Karakas et al. 2011; Karim et al. 2011; Kebebew and Shibeshi 2013; Klodzinska, Tatarczynska, Stachowicz, et al. 2004; Klodzinska,

Tatarczynska, Chojnacka-Wojcik, et al. 2004; Kong et al. 2006; Kumar and Bhat 2014; Kurhe et al. 2014; Kuribara et al. 2000; LaBuda and Fuchs 2001; Langen et al. 2005; Leggio et al. 2011; Lepicard et al. 2000; Jie Liu et al. 2015; Lolli et al. 2007; Mahendra and Bisht

2011; Mansouri et al. 2014; Martinez et al. 2006; Mechan et al. 2002; de Melo et al. 2006; Melo et al. 2010; Mesfin, Asres, and Shibeshi 2014; Meyer et al. 2013; Mi et al. 2005; Micale et al. 2009; Micale et al. 2008; Molander et al. 2011; Molina-Hernandez et al. 2004; Mora et

al. 2005; Moreira et al. 2014; Nagaraja et al. 2012; Ochoa-Sanchez et al. 2012; Ognibene et al. 2008; Okuyama et al. 1999; Onusic et al. 2002; Pain et al. 1999; Paine, Jackman, and Olmstead 2002; Parent et al. 2012; Pellow et al. 1985; la Pena et al. 2013; Peng et al. 2004;

Pires et al. 2013; Plaznik et al. 1994; Ponten et al. 2011; Popik et al. 2006; Radulovic et al. 2013; Rago et al. 1988; Ramanathan, Jaiswal, and Bhattacharya 1998; Raquibul Hasan et al. 2009; Rejon-Orantes et al. 2013; Rex, Morgenstern, and Fink 2002; Rochford et al. 1997;

Saiyudthong and Marsden 2011; Sakaue et al. 2003; Santos Rosa et al. 2012; Satyan et al. 1998; Schmitt, Luddens, and Hiemke 2002; Schmitt, Luddens, and Hiemke 2001; Sherif et

al. 1994; Da Silva et al. 1996; Silva et al. 2007; Simpson and Kelly 2012; Sorra et al. 2014; de Sousa et al. 2007; Srinivasan, Suresh, and Ramanathan 2003; Stankevicius et al. 2008; Stefanski et al. 1992; Steiner, Lecourt, and Jenck 2012; Stemmelin et al. 2008; Sugiyama et

al. 2012; Swami et al. 2014; Taiwo et al. 2012; Tanaka, Satou, and Koike 2013; Tatarczynska et al. 2004; Thippeswamy et al. 2011; Thompson, Grabowski-Boase, and Tarantino 2015; Thongsaard et al. 1996; Tolardo et al. 2010; Varty et al. 2002; Venancio et

al. 2011; Volke et al. 1998; Wada and Fukuda 1991; Wanasuntronwong et al. 2012; Wang et al. 2015; Wesolowska and Nikiforuk 2007; Wikinski et al. 2001; Wolfman et al. 1994; Yadav,


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Kawale, and Nade 2008; Yamada et al. 2000; Yao et al. 2010; Yasumatsu et al. 1994; Zanoli et al. 2002; L.-M. Zhang et al. 2014; Zheng et al. 2009). Calculation of an average Hedges’ g

(Cumming 2012) for 386 experiments revealed that diazepam had a very large effect on ARDEB, with a -1.26 g [95CI -1.36, -1.17] reduction compared with untreated control animals (Figure 3, Table 2). However, as Egger’s regression indicated the source literature was affected by publication bias, trim-and-fill correction indicated a smaller, but still large, effect of -0.85 g [95CI -0.74, -0.96]. The meta-analysis had a moderate level of heterogeneity, indicating that the assay type, laboratory, dosage, species, strain and other possible sources of experimental variation play a role in this literature.

5-HT1A receptor function influences ARDEB

Following negative publicity regarding the adverse effects of benzodiazepines (Tone 2009), pharmaceutical companies focused on the serotonergic system (Griebel and Holmes 2013). Of the fourteen mammalian serotonergic receptors, the serotonin receptor 5-HT1A has been targeted for its proposed connection with anxiety disorders and depression (Samuels et al. 2014). More than 1200 articles describe experiments connecting 5-HT1A agonism with rodent anxiety (Griebel and Holmes 2013). However, a substantial proportion of those articles reported that 5-HT1A agonists or knockout of the Htr1a gene either produced no effect on anxiety or an effect that was opposite to the receptor’s proposed mode of action

(Griebel and Holmes 2013). We performed systematic reviews of gene manipulations of Htr1a and identified 11 knockout articles (Piszczek et al. 2013; Vinkers et al. 2010; Gleason

et al. 2010; Groenink et al. 2003; Klemenhagen et al. 2006; Freeman-Daniels, Beck, and Kirby 2011; A. Jain et al. 2012; Ramboz et al. 1998; Ferrés-Coy et al. 2013; Parks et al.

1998; Gross et al. 2002). Meta-analysis of the knockout data revealed that removal of Htr1a produced a moderate increase (Hedges’ g = 0.73 [95CI 0.50, 0.96] , p = 3.5 x 10-10) in ARDEB phenotypes (Figure 4A). The three studies of Htr1a overexpression found by the review (Kusserow et al. 2004; Bert et al. 2006; Audero et al. 2013) indicated that this

intervention moderately decreased ARDEB (g = -0.6 [95CI -1.3, 0.13], p = 0.11; Figure 4B). The cumulative sample size of Htr1a overexpression is large (N = 100, 98), though the moderate effect size was not statistically significant and the heterogeneity was high (I2 = 80%). These results confirm that Htr1a function has a moderate effect on rodent anxiety.

Anxiotropic effects of the serotonin transporter

The serotonin transporter (SERT) is the target for the selective serotonin reuptake inhibitors (SSRIs), a class of drugs used to treat depression and anxiety (Baldwin et al. 2014). Meta-


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analysis of thirteen knockout studies (Holmes, Yang, et al. 2003; Holmes, Lit, et al. 2003; Lira et al. 2003; Li et al. 2004; Carroll et al. 2007; Kalueff et al. 2007; Olivier et al. 2008; Line

et al. 2011; Schipper et al. 2011; Zhao et al. 2006; Kalueff, Jensen, and Murphy 2007; Moya et al. 2011; Pang et al. 2011) revealed a large anxiogenic effect (g = 0.88 [95CI 1.26, 0.23] , p = 5.2 x 10-14; Figure 5A) produced by knocking out the SERT gene, Htt. However, a funnel

plot and Egger’s regression revealed a pronounced bias in reported effect sizes (Egger’s test p = 6.7 X 10-6, Table 2). Trim-and-fill adjustment filled the left segment of the funnel plot with ten imputed data points so as to obtain a symmetric funnel plot, reducing the effect size to g = 0.57 [95CI 0.29, 0.86], a moderate effect. Only two articles studying the effect of Htt overexpression on ARDEB were found (Jennings et al. 2006; Line et al. 2011). Meta-analysis revealed a large anxiolytic effect (g = -0.94 [95CI -1.69, -0.20], p = 0.013; Figure 5B) in EPM and OF assays (no LD articles were found). The transporter gene knockout and overexpression effects clearly connect Htt function to rodent anxiety. However, the

direction of effects is the opposite of what would be expected from the clinical application of SERT inhibitors, given that SSRI reduction of SERT function is thought to have a therapeutic, anxiety-reducing effect.

The effects of environmental stressors in ARDEB

Environmental stressors have physiological effects on animals that promote the anxiety-like state (van Praag 2003). To survey a range of stress modalities we selected acute pain, bodily restraint and social isolation for review; all three have been found to promote anxiety in humans (Sherif and Oreland 1995).

The systematic review identified seven papers measuring the effect of acute pain on ARDEB (Yan Liu et al. 2015; Matsuzawa-Yanagida et al. 2008; Schellinck, Stanford, and Darrah 2003; Benbouzid et al. 2008; Parent et al. 2012; Leite-Almeida et al. 2012; Shang et

al. 2014). Meta-analysis of the 21 experiments therein indicated a moderate anxiogenic effect (g = 0.56 [95CI 0.19, 0.93], p = 2.9 x 10-3; Figure 6A).

Review of 16 studies of rodent bodily restraint (Anand, Gulati, and Ray 2012; Busnardo et al. 2013; Carvajal et al. 2004; Chesworth et al. 2012; Estanislau and Morato 2005; Granjeiro et al. 2011; Harris et al. 2001; Joshi, Ray, and Gulati 2014; Jing Liu et al.

2011; Locchi et al. 2008; Lunga and Herbert 2004; Nosek et al. 2008; Ouagazzal et al. 2003; D. G. Reis et al. 2011; Rylkova et al. 2009; Walf and Frye 2012) containing 21 experiments indicated that it had an overall moderate anxiogenic effect in EPM and OF assays (g = 0.70 [ 95% CI 0.82 - 1.32], p = 0.027; Figure 6B). The restraint meta-analysis had a high level of


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heterogeneity, I2 = 89%; a subgroup analysis by assay type revealed that the different assays were not the source of this variability (data not shown).

The systematic review identified 50 articles on social isolation and ARDEB (Quintino-dos-Santos et al. 2014; Pisu et al. 2013; Linge, Pazos, and Diaz 2013; Xiao Liu et al. 2013; Yorgason et al. 2013; Chappell et al. 2013; Cuenya et al. 2012; Carrier and Kabbaj 2012; F.

M. C. V. Reis et al. 2012; Y. Zhang et al. 2012; Yildirim, Erol, and Ulupinar 2012; Djordjevic et al. 2012; Workman et al. 2011; Hermes et al. 2011; Conrad et al. 2011; Bledsoe et al. 2011; Pisu et al. 2011; Kokare et al. 2010; Santos, de Andrade, and Graeff 2010; Ryu et al.

2009; H. Koike et al. 2009; Doremus-Fitzwater, Varlinskaya, and Spear 2009; McCool and Chappell 2009; Brenes, Padilla, and Fornaguera 2009; Lukkes et al. 2009; Imanaka et al. 2008; Leussis and Andersen 2008; Pritchard et al. 2008; Wei et al. 2007; Knuth and Etgen

2007; Estelles et al. 2007; Imanaka et al. 2006; Blakley and Pohorecky 2006; Thorsell et al. 2006; Hirani et al. 2005; Voikar et al. 2005; Abramov et al. 2004; Moragrega et al. 2003;

Majercsik et al. 2003; Blednov et al. 2001; Lapiz et al. 2001; Cheeta, Irvine, and File 2001; Wright and Ingenito 2001; Haller, Halász, and Makara 2000; Serra et al. 2000; Haller and Halász 1999; Da Silva et al. 1996; Fone et al. 1996; Rodgers and Cole 1993; Das et al.

2014). Meta-analysis revealed a small anxiogenic effect (g = 0.33 [95CI 0.21, 0.44], p = 3.4 x 10-8; Figure 6C). However, Egger’s regression revealed funnel plot asymmetry, and the trim-and-fill method corrected the anxiogenic effect to only 0.21 g [95CI 0.07, 0.34], p = 3.1 x 10-3) a very small anxiotropic effect (Figure 2B). It appears that, unlike the physical stressors, social isolation has only a modest influence on the ARDEB assays.

The role of CRH and its receptor on ARDEB

Several neuropeptide-related genes involved in stress signaling have been linked to anxiety, notably the peptide, corticotropin-releasing hormone (CRH; also known as corticotropin-releasing factor) (Kormos and Gaszner 2013) and its receptor, CRHR1. Two studies that examined the effects of Crh knockouts on ARDEB were found (Weninger et al. 1999), which revealed only a small effect (g = 0.30 [95CI -0.32, 0.92], p = 0.34; Figure 7A). This supports the idea that CRH has only a modest effect on the ARDEB; however, the meta-analytic result may suffer from insufficient precision as the cumulative sample size was only (N = 20, 21).

CRH exerts its biological action via two receptors known as CRHR1 and CRHR2. The two receptors are pharmacologically distinct and only the former has been widely studied in the context of anxiety (Owens and Nemeroff 1991; Paez-Pereda, Hausch, and


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Holsboer 2011). Meta-analysis (Liebsch et al. 1995; Liebsch et al. 1999; Smith et al. 1998; Müller et al. 2003; Gammie and Stevenson 2006; Trimble et al. 2007) found that, in contrast

to the Crh knockout, deletion of Crhr1 had a large anxiolytic effect on ARDEB (g = -1.0 [95CI -1.30, -0.70], p = 6.64 x 10-11; Figure 7B). The discordance between Crh and Crhr1 knockout effects has previously been attributed to the action of other peptide ligand(s) of Crhr1, either urocortin or another, unidentified ligand (A. J. Dunn and Swiergiel 1999).

Genes with no purported effect on anxiety

Some of the interventions in the systematic review were found to have modest effects on ARDEB. We aimed to contextualize the magnitude of proposed anxiety factors with factors that the literature is not connected with anxiety. Such factors are not generally mentioned in anxiety reviews and it is difficult to identify them with database searches as they are less likely to be published or, when published, are unlikely to be the focus of a study and thus may not be mentioned in the abstract. We conducted an ad hoc search of the PubMed and EMBASE databases for systems unimportant to anxiety, which identified adenylyl cyclase 1

(Storm et al. 1998; Krishnan et al. 2008) and synaptotagmin 1 (Powell et al. 2004) as being

proposed to have no anxiety-related activity. Meta-analyses confirmed that both interventions have very small (Hedges’ g < 0.3) effects on ARDEB (Figure 8).


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DISCUSSION

Summary of evidence

Inspection of the forest plots reveals that all of the primary publication sets include experimental effect sizes that are discordant, either in direction (anxiolytic versus anxiogenic) and/or magnitude. The generality of discordance in the literature emphasizes the utility of meta-analysis to behavioral neuroscience to give a quantitative overview and to synthesize the best evidence available. Of ten analyses of putative anxiotropic interventions, eight yielded at least moderate meta-analytic effect sizes and two produced small effect sizes (Figure 9). Of the moderate effect size factors, one (Htr1a overexpression) had a non-statistically significant test result. However, the majority of putative anxiety-related genes do have at least moderate effects on the rodent defense behaviors. The synthetic data strongly confirm that diazepam, the serotonergic system, environmental stressors, and Crhr1 influence an anxiety-like process in the mouse brain.

Limitations

Some limitations of this study include that only published data were used and that only English language articles were used. Some studies had to be excluded from the meta-analysis during the full text scan because they did not report measures of variance. Only those studies were selected for meta-analysis which report time or percent time spent in exposed arena could be selected for meta-analysis. It is possible the inaccessible may affect the summary measures when added to the meta-analyses. The presence of publication bias in the three largest data sets assessed for bias, suggests that inclusion of further (e.g. unpublished) data to the smaller meta-analyses would be expected, on average,to lower these effect sizes as well. Heterogeneity was at least moderate (I2 > 50%) in five of the meta-analyses, indicating that the random effect model is insufficient to explain the variance in these data sets. Future work will include the implementation of mixed regression models that try to account for all or more of the unexplained variance.

Knowledge gaps, publication bias and heterogeneity

We found no knowledge gaps per se, as all the purported anxiety-related factors had at least two studies. Nevertheless, Htt overexpression, Crh knockouts and the non-anxiety

genes had total experimental iterations below N = 64, 64, the sample size needed for 80% power to detect a 0.5 standard deviation effect size, indicating that more work could be done here. Of the six factors for which ≥ 20 studies were published, three were affected by


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publication bias. Heterogeneity was high or moderate for five of the factors, and low for the other five factors reviewed, indicating that laboratory, strain, assay type and other protocol variations played a variable role across factors.

Htr1a interventions have weaker ARDEB effects than diazepam

Meta-analysis of Htr1a knockout and overexpression revealed that both have moderate anxiotropic effects. Both effects were smaller (0.73 and -0.6 g respectively) than the effect

produced by diazepam (-0.85 g) suggesting that compounds aiming to increase 5-HT1A function may be a poor strategy to reduce anxiety, despite the pronounced Htr1a knockout

outcome. This view is supported by recent clinical meta-analyses that have concluded that drugs targeting 5-HT1A - the azapirones - appear inferior to benzodiazepines for generalized anxiety disorder (Chessick et al. 2006) and that there is insufficient evidence to support azapirone use in panic disorder (Imai et al. 2014).

The Htt-SSRI paradox in defense behaviors

Drugs that target SERT, the SSRIs, are recommended as the first line of pharmacological

treatment for anxiety (Baldwin et al. 2014). Blocking SERT-mediated transport (reuptake) of serotonin from the synaptic cleft is the proposed mechanism of SSRI anxiety reduction, although rodent studies of chronic SSRI effects on ARDEB have been inconclusive (Perez-Caballero et al. 2014; Griebel and Holmes 2013). Given the the inhibitors’ clinical use, it is surprising that Htt knockouts have elevated anxiety relative to controls (0.57 g) and that Htt

overexpression reduces rodent anxiety (-0.94 g).The reason for this discordance is not clear. In some cases the authors of the primary Htt knockout studies have not discussed this paradox (Moya et al. 2011; Schipper et al. 2011; Carroll et al. 2007; Kalueff et al. 2007).

Other authors have remarked that the underlying reason remains unclear (Lira et al. 2003; Holmes, Yang, et al. 2003) or have called the validity of ARDEB assays into doubt (Pang et al. 2011). Primary study authors have also proposed two explanatory hypotheses. The first

is that increased anxiety arises from developmental alterations present in Htt knockouts not present in chronically drug-treated animals (Olivier et al. 2008; Zhao et al. 2006; Holmes,

Yang, et al. 2003). This hypothesis could be tested with conditional knockdown models, i.e. in animals with Htt only deleted at the adult stage. While systematic review of PubMed and EMBASE did not identify any published reports of post-developmental Htt knockout

experiments (e.g., using floxed Htt), researchers have analyzed the anxiety-related effects of conditionally ablating the Pet-1 gene. Pet-1 is a transcription factor with an expression range that overlaps closely with the expression of Htt. In mice with Pet-1 removed in


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adulthood, mRNA levels of Htt are substantially reduced (Chen Liu et al. 2010). Like Htt knockouts, these mice show increased anxiety-like behaviors in multiple ARDEB assays (Chen Liu et al. 2010), eroding confidence in the developmental alteration hypothesis. A

second hypothesis to explain the Htt/SSRI paradox is that there is a J-shaped relationship between Htt function and anxiety, i.e., both wild-type and knockout animals would have higher anxiety relative to animals with intermediate function (Olivier et al. 2008). Further

study is required to fully understand the role of Htt and SSRIs in ARDEB.

Conclusions

This study supports the hypothesis that some mainstream anxiety-related interventions have trivial or unconfirmed effects in rodent defense behavior assays. However, this study also confirms a drug, two environmental stressors and a number of genes as authentic ARDEB influencers. These verified anxiety-related interventions (diazepam, Htr1a gene

knockout, Htt gene knockout, Htt gene overexpression, acute pain, restraint and Crhr1 gene knockout) can be used as reference manipulations to validate other animal models of anxiety-like behavior.

Meta-analysis has the ability to aggregate information and resolve discordance in the primary literature. This ability may be particularly useful in behavioral neuroscience where most primary articles describe experiments with low power (or precision) (Button et al. 2013). Precise estimation of effect magnitude is important both to build quantitative

models of brain function and to improve the ability of preclinical studies to predict clinical efficacy.


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Author Contributions

FM performed data extraction, systematic review, and meta-analyses and co-wrote the

manuscript. CLL, JHW, DJJP and BL performed systematic review, and data extraction. JH did meta-analyses and publication bias analysis, performed data extraction checks, and contributed writing. ACC designed the study, guided the work, and wrote the manuscript. All authors were involved in the revision of the draft manuscript and have agreed to the final content.

Funding Information

The authors were supported by Biomedical Research Council block grants to the Neuroscience Research Partnership and the Institute of Molecular and Cell Biology. FM and ACC also received support from Duke-NUS Graduate Medical School. JH received support from the A*STAR Graduate Academy. FM and ACC received additional support from a Nuffield Department of Medicine Fellowship and a Wellcome Trust block grant to the University of Oxford. ACC also received support from A*STAR Joint Council Office grant 1231AFG030 and NARSAD Young Investigator Award 17741. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.


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FIGURES AND TABLES

Figure 1. Flow chart of the systematic literature review of 10 anxiotropic interventions.

The literature was reviewed in a four-stage process, starting with searches of the Pubmed and EMBASE databases that yielded 1156 articles, followed by three screens of increasing detail, reviewing the article title, abstract, and full text for experimental design. A total of 298 articles were used in the meta-analysis. Further details are given in Table 1 and the Methods section.

Figure 2. Funnel plots of three meta-analyses with evidence for publication bias.

Where at least ten experiments were available for meta-analysis, the effect sizes (Hedges’ g) of the experiments are plotted against their respective standard errors. Points on each plot represent individual experiments. The triangle bounded by dotted lines indicates the area where 95% of studies are expected to fall, in the absence of both publication bias and

study heterogeneity. Shown here are funnel plots for experiments on (A) diazepam, (B) social isolation, and (C) Htt knockout.

Figure 3. Meta-analysis of diazepam on rodent anxiety related behavior.

Meta-analysis of rodent diazepam effect sizes, shown as a forest plot of standardized effect sizes (Hedges’ g). The meta-analysis is sub-grouped by animal species. Error bars indicate the 95% confidence intervals of standardized mean difference. The weighted average mean effect size of subgroups and all studies is represented by the central vertices of a red diamond; the outer vertices indicate the 95% confidence intervals. Control and treatment samples sizes are given in the columns listed as NC and NT respectively.

Figure 4. Meta-analyses of serotonin receptor 1A interventions on rodent anxiety-

related behaviors.

Meta-analysis of effect sizes of serotonin-targeted interventions is shown as a forest plot of standardized effect sizes (Hedges’ g). Error bars indicate the 95% confidence intervals of g. The weighted average mean effect size of all studies is represented by the central vertices of a red diamond; the outer vertices indicate the 95% confidence intervals. Control and treatment samples sizes (NC, NT) and the assay types of the studies are given; elevated plus maze (EPM), open field (OF) and light-dark box (LD). Effects of: A. Serotonin receptor gene Htr1a knockout models. B. Htr1a overexpression.


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Figure 5. Meta-analyses of serotonin transporter interventions on rodent anxiety-

related behaviors.

Meta-analysis of effect sizes of serotonin-targeted interventions is shown as a forest plot of standardized effect sizes (Hedges’ g). Error bars indicate the 95% confidence intervals of g. The weighted average mean effect size of all studies is represented by the central vertices of a red diamond; the outer vertices indicate the 95% confidence intervals. Control and treatment samples sizes (NC, NT) and the assay types of the studies are given; elevated plus maze (EPM), open field (OF) and light-dark box (LD). Effects of: A. Serotonin transporter gene (Htt) knockout models B. Htt overexpression models.

Figure 6. Meta-analyses of experiments on the stress-anxiety relationship in rodents.

Meta-analysis of effect sizes of stress-anxiety interventions, shown as a forest plot of standardized effect sizes (Hedges’ g). Error bars indicate the 95% confidence intervals of g.

The weighted average mean effect size of all studies is represented by the central vertices of a red diamond; the outer vertices indicate the 95% confidence intervals. Control and treatment samples sizes (NC, NT) and the assay types of the studies are given; elevated plus maze (EPM), open field (OF) and light-dark box (LD). Effects of: A. Acute pain. B. Restraint stress (immobilization). C. Social isolation.

Figure 7. Meta-analyses of the effects of stress signaling genes on anxiety-related

behaviors.

Meta-analysis of effect sizes of stress signaling genes, shown as a forest plot of standardized effect sizes (Hedges’ g). Error bars indicate the 95% confidence intervals of g.

The weighted average mean effect size of all studies is represented by the central vertices of a red diamond; the outer vertices indicate the 95% confidence intervals. Control and treatment samples sizes (NC, NT) and the assay types of the studies are given; elevated plus maze (EPM), open field (OF) and light-dark box (LD). Effects of: A. Crh gene knockout models. B. Crhr1 gene knockout models.

Figure 8. Meta-analyses of interventions known to be unrelated to anxiety.

Meta-analysis of effect sizes of non-anxiety-related interventions, shown as a forest plot of standardized effect sizes (Hedges’ g). Error bars indicate the 95% confidence intervals of g. The weighted average mean effect size of all studies is represented by the central vertices of a red diamond; the outer vertices indicate the 95% confidence intervals. Control and treatment samples sizes (NC, NT) and the assay types of the studies are given; elevated plus


https://doi.org/10.1101/020701


46

maze (EPM), open field (OF) and light-dark box (LD). Effects of: A. Adcy1 gene knockout models. B. Synaptotagmin gene (Syt1) mutation models.

Figure 9. Summary effect sizes of all meta-analyses.

The weighted mean effect sizes of all 12 interventions are shown here. Each mean effect size is represented by the central vertices of a diamond; the outer vertices indicate the 95% confidence intervals. The horizontal axis is Hedges’ g, the standard deviation change

relative to control animals. Color indicates direction (green = anxiolytic, red = anxiogenic) and statistical significance (grey = statistically non-significant). The diamonds for the diazepam, social isolation, and Htt KO meta-analyses represent the summary effect sizes after trim-and-fill bias correction.

Table 1. Summary of systematic reviews of anxiety-related interventions in mouse and

rat.

The PubMed and Embase query phrases used to identify articles that might contain data relevant to the interventions and assays of interest are detailed. Title, abstract and full-text searches were performed to identify articles meeting the selected criteria. Where there were fewer than fifteen articles meeting the selection criteria, all relevant data in all articles found were used for data extraction and meta-analysis. Where there were more articles, we took a random sample of fifteen papers and used only relevant data therein for meta-analysis.

Table 2. Results of Egger’s linear regression test for funnel plot asymmetry across six

meta-analyses

Where at least ten experiments were available for meta-analysis, Egger’s linear regression test for funnel plot asymmetry was performed. For each meta-analysis, the number of studies included, the vertical intercept of the linear regression, the corresponding 95% confidence interval for the intercept, and the p-value of Egger’s test are listed.

Table 3. Characteristics of included experiments.

Characteristics of experiments from included studies are listed with Pubmed ID, year of study, and figure panel. The assay type, assay duration, variable used in experiment, route of injection, drug dosage, treatment duration, species, strain and gender are also detailed. Assay duration and treatment duration are listed in minutes. Dosage is listed in mg per kg body weight of animal. Cells containing NA indicate that the information is not available for the underlying intervention.


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47

Supporting Information Legends

Supporting Information 1. Spreadsheet containing extracted data (.csv file)

Supporting Information 2. Extracted meta-analytic data in R-compatible format

(.RData file)

Supporting Information 3. R markdown code (.Rmd) used for meta-analysis and

plotting


https://doi.org/10.1101/020701


Figure 1. Flow chart of the systematic literature review of 10 anxiotropic interventions. The literature was reviewed in a four-stage process, starting with a PubMed search that yielded 1156 articles, followed by three screens of increasing detail, reviewing the article title, abstract, and full text for experimental design. A total of 297 articles were used in the meta-analysis. Further details are given in Table 1 and the Methods section.

1155 articles identified in PubMed and Embase searches for 10 anxiety related interventions using the search phrases given in Table1

500 excluded

655 articles included

Title Screen

Abstract Screen 149 excluded

506 articles included

Full text Screen 224 excluded

282 articles included in meta-analyses


https://doi.org/10.1101/020701


Diazepam

Sta

ndar

d E

rror

2.0

1.0

0.0

-5.0 0.0 5.0

Uncorrected: g = -1.2695% CI [-1.36; -1.17]

Corrected: g = -0.8595% CI [-0.96; -0.74]

Isolation

Hedges' g

0.6

0.3

0.0

-2.0 -1.0 0.0 1.0 2.0 3.0

Uncorrected: g = 0.3395% CI [0.21; 0.44]Corrected: g = 0.21

95% CI [0.07; 0.34]

HTT KO

0.9

0.4

0.0

-1.0 0.0 1.0 2.0

Uncorrected: g = 0.8895% CI [0.65; 1.1]

Corrected: g = 0.5795% CI [0.29; 0.86]

Sta

ndar

d E

rror

Sta

ndar

d E

rror

Hedges' g

Hedges' g

Figure 2. Funnel Plots for Three Meta-Analysis With Evidence of Publication Bias.Where at least ten experiments were available for meta-analysis, the effect sizes (Hedges’ g) of the experiments are plotted against their respective standard errors. Points on each plot represent individual experiments. The triangle bounded by dotted lines indicates the area where 95% of studies are expected to fall, in the absence of both publication bias and study heterogeneity. Shown here are funnel plots for experiments on (A) diazepam, (B) social isolation, and (C) Htt knockout.

A

C

B


https://doi.org/10.1101/020701


Diazepam

Random e ects modelHeterogeneity: I^2=67.2%, Tau^2=0.5425, p<0.0001

Species = Mice

Species = Rats

Random e ects model

Random e ects model

Heterogeneity: I^2=74%, Tau^2=0.8632, p<0.0001

Heterogeneity: I^2=53.9%, Tau^2=0.279, p<0.0001

González-Trujano ME 2012 Mice 0.01mg/kg MaleGonzález-Trujano ME 2012 Mice 0.03mg/kg MaleGonzález-Trujano ME 2015 Mice 0.1mg/kg MaleLeggio 2011 Mice 0.1mg/kg MaleGonzález-Trujano ME 2012 Mice 0.1mg/kg MalePonten 2011 Mice 0.1mg/kg MaleLeggio 2011 Mice 0.1mg/kg MaleLeggio 2011 Mice 0.1mg/kg MaleLeggio 2011 Mice 0.1mg/kg MaleJones GH 1994 Mice 0.2mg/kg MaleEne HM 2015 Mice 0.25mg/kg MaleGonzález-Trujano ME 2012 Mice 0.3mg/kg MaleWolfman C 1994 Mice 0.3mg/kg MaleAssié MB 1993 Mice 0.3mg/kg MaleJones GH 1994 Mice 0.39mg/kg MaleMahendra P 2011 Mice 0.5mg/kg MaleLeggio 2011 Mice 0.5mg/kg MaleLeggio 2011 Mice 0.5mg/kg MaleLeggio 2011 Mice 0.5mg/kg MaleOgnibene E 2008 Mice 0.5mg/kg MaleEne HM 2015 Mice 0.5mg/kg MalePonten 2011 Mice 0.5mg/kg MalePeng WH 2004 Mice 0.5mg/kg MaleMes n M 2014 Mice 0.5mg/kg MaleLepicard EM 2000 Mice 0.5mg/kg MaleLepicard EM 2000 Mice 0.5mg/kg MaleRochford J 1997 Mice 0.5mg/kg MaleSakaue M1 2003 Mice 0.5mg/kg MaleLeggio 2011 Mice 0.5mg/kg MaleWolfman C 1994 Mice 0.6mg/kg MaleBaretta IP 2012 Mice 0.75mg/kg MaleTolardo R 2010 Mice 0.75mg/kg MaleRägo L 1988 Mice 0.75mg/kg MaleJones GH 1994 Mice 0.78mg/kg MaleBrioni 1994 Mice 0.997mg/kg MaleMicale 2009 Mice 1mg/kg MaleMartínez AL 2006 Mice 1mg/kg MaleLeggio 2011 Mice 1mg/kg MaleLeggio 2011 Mice 1mg/kg MaleGonzález-Trujano ME 2012 Mice 1mg/kg MaleKarim N 2011 Mice 1mg/kg MaleSilva 2007 Mice 1mg/kg MaleMelo FH 2010 Mice 1mg/kg MaleYadav AV 2008 Mice 1mg/kg MaleThippeswamy BS 2011 Mice 1mg/kg MaleLolli LF 2007 Mice 1mg/kg MaleLolli LF 2007 Mice 1mg/kg MalePeng WH 2004 Mice 1mg/kg MaleFajemiroye JO 2014 Mice 1mg/kg MaleGirish C 2013 Mice 1mg/kg MaleGirish C 2013 Mice 1mg/kg MaleMicale 2008 Mice 1mg/kg MaleConsoli D 2007 Mice 1mg/kg MaleNagaraja TS 2012 Mice 1mg/kg MaleGirish C 2013 Mice 1mg/kg MaleMicale 2009 Mice 1mg/kg Malede Sousa FC 2007 Mice 1mg/kg MaleGomes PB 2010 Mice 1mg/kg MaleBahi A 2014 Mice 1mg/kg MaleBlainski A 2010 Mice 1mg/kg MaleWang X 2015 Mice 1mg/kg MaleNagaraja TS 2012 Mice 1mg/kg MaleRejón-Orantes JC 2013 Mice 1mg/kg Malede Sousa FC 2007 Mice 1mg/kg MaleKebebew Z 2013 Mice 1mg/kg MalePonten 2011 Mice 1mg/kg Malede Melo CT 2006 Mice 1mg/kg MaleSorra K 2014 Mice 1mg/kg MaleRochford J 1997 Mice 1mg/kg MaleLepicard EM 2000 Mice 1mg/kg MaleKuribara 2000 Mice 1mg/kg MaleRaquibul Hasan SM 2009 Mice 1mg/kg MaleBhattacharya 1991 Mice 1mg/kg MaleCole 1995 Mice 1mg/kg MaleLepicard EM 2000 Mice 1mg/kg MaleHui 2002 Mice 1mg/kg MaleSakaue M1 2003 Mice 1mg/kg MaleChen 2004 Mice 1mg/kg MaleWanasuntronwong 2012 Mice 1mg/kg MaleFernandez SP 2008 Mice 1mg/kg MaleLeggio 2011 Mice 1mg/kg MaleLeggio 2011 Mice 1mg/kg MaleDalvi A 2001 Mice 1.5mg/kg MaleRägo L 1988 Mice 1.5mg/kg MaleDalvi A 1999 Mice 1.5mg/kg MaleJones GH 1994 Mice 1.56mg/kg MaleGonzález-Trujano ME 2012 Mice 2mg/kg MaleGupta D 2014 Mice 2mg/kg MaleKurhe YV 2014 Mice 2mg/kg MaleCosta JP 2014 Mice 2mg/kg Malede Almeida 2012 Mice 2mg/kg MaleKarim N 2011 Mice 2mg/kg MaleSwami SU 2014 Mice 2mg/kg MaleFortes AC 2013 Mice 2mg/kg MaleBhatt S 2013 Mice 2mg/kg MaleHan H 2009 Mice 2mg/kg MaleLepicard EM 2000 Mice 2mg/kg MaleYao Y 2010 Mice 2mg/kg MaleRochford J 1997 Mice 2mg/kg MaleKarim N 2011 Mice 2mg/kg MaleChen SW 2005 Mice 2mg/kg Malede la Peña JB 2013 Mice 2mg/kg MaleFernandez SP 2008 Mice 2mg/kg MaleKarim N 2011 Mice 2mg/kg MaleChen SW 2005 Mice 2mg/kg MaleMi XJ 2005 Mice 2mg/kg MaleZhang LM 2014 Mice 2mg/kg MaleColla AR 2015 Mice 2mg/kg MaleCha HY 2005 Mice 2mg/kg MaleCha HY 2005 Mice 2mg/kg MaleCha HY 2005 Mice 2mg/kg MaleCha HY 2005 Mice 2mg/kg MaleCha HY 2005 Mice 2mg/kg MaleLiu J 2015 Mice 2mg/kg MaleLepicard EM 2000 Mice 2mg/kg MaleWanasuntronwong 2012 Mice 2mg/kg MaleSakaue M1 2003 Mice 2mg/kg MaleLiu J 2015 Mice 2mg/kg MaleBoerngen-Lacerda 2000 Mice 2mg/kg MaleHarro J 1990 Mice 2.5mg/kg MaleJones GH 1994 Mice 3.13mg/kg MaleLarissa FdA 2013 Mice 5mg/kg MaleJones GH 1994 Mice 6.25mg/kg MaleTanaka M 2013 Mice 10mg/kg MaleJones GH 1994 Mice 12.5mg/kg Male

Harada K 2006 Rats 0.1mg/kg MaleGriebel G 1999 Rats 0.1mg/kg MaleGriebel G 1998 Rats 0.25mg/kg MaleYasumatsu H 1994 Rats 0.25mg/kg MaleGriebel G 1999 Rats 0.3mg/kg MaleDunn 1998 Rats 0.3mg/kg MaleHarada K 2006 Rats 0.32mg/kg MaleBrioni 1994 Rats 0.484mg/kg MaleSrinivasan J 2003 Rats 0.5mg/kg MaleYasumatsu H 1994 Rats 0.5mg/kg MaleDrapier D 2007 Rats 0.5mg/kg MaleGriebel G 1999 Rats 0.5mg/kg MaleJain 2005 Rats 0.5mg/kg MaleGriebel G 1998 Rats 0.5mg/kg MaleDa Silva NL 1996 Rats 0.75mg/kg MaleBrioni 1994 Rats 0.997mg/kg MaleSugiyama A 2012 Rats 1mg/kg MaleSaitoh A 2013 Rats 1mg/kg MaleCarro-Juárez M 2012 Rats 1mg/kg MaleVenâncio ET 2011 Rats 1mg/kg MaleOchoa-Sanchez R 2012 Rats 1mg/kg MaleSaiyudthong 2011 Rats 1mg/kg MaleHarada K 2006 Rats 1mg/kg MaleBarbosa PR 2008 Rats 1mg/kg MaleMansouri MT 2014 Rats 1mg/kg MaleMansouri MT 2014 Rats 1mg/kg Malede-Paris F 2000 Rats 1mg/kg MaleCechin EM 2003 Rats 1mg/kg MaleTaiwo AE 2012 Rats 1mg/kg MaleSugiyama A 2012 Rats 1mg/kg MaleDunn 1998 Rats 1mg/kg MaleTaiwo AE 2012 Rats 1mg/kg MaleKalouda T 2015 Rats 1mg/kg MaleTaiwo 2012 Rats 1mg/kg MaleThongsaard W 1996 Rats 1mg/kg MaleYasumatsu H 1994 Rats 1mg/kg MaleHasenöhrl RU 1996 Rats 1mg/kg MaleMechan AO 2002 Rats 1mg/kg MalePellow S 1985 Rats 1mg/kg MaleParent AJ 2012 Rats 1mg/kg MaleThongsaard W 1996 Rats 1mg/kg MaleGriebel G 1999 Rats 1mg/kg MalePaine TA 2002 Rats 1mg/kg MaleMechan AO 2002 Rats 1mg/kg MaleFrassetto 2010 Rats 1mg/kg MaleFaria 1997 Rats 1mg/kg MaleGriebel G 1998 Rats 1mg/kg MaleGriebel G 2002 Rats 1mg/kg MaleJászberényi M 2007 Rats 1mg/kg MaleGriebel G 1999 Rats 1mg/kg MaleOkuyama S 1999 Rats 1mg/kg MaleDa Silva NL 1996 Rats 1mg/kg MaleJaszberenyi 2009 Rats 1mg/kg MaleSteiner MA 2012 Rats 1mg/kg MaleMeyer L 2013 Rats 1.25mg/kg MaleDunn RW 1989 Rats 1.25mg/kg MaleMeyer L 2013 Rats 1.25mg/kg MaleKlodzinska A 2004 Rats 1.25mg/kg MaleKlodzinska 2004 Rats 1.25mg/kg MaleTatarczy ska E 2004 Rats 1.25mg/kg MaleWeso owska A 2007 Rats 1.25mg/kg MaleJastrz bska-Wi sek M 2014 Rats 1.25mg/kg MaleSimpson J 2012 Rats 1.25mg/kg MaleMolina-Hernández M 2004 Rats 1.3mg/kg MaleZanoli 2002 Rats 1.5mg/kg MaleGriebel G 1997 Rats 1.5mg/kg MaleSherif F 1994 Rats 1.5mg/kg MaleDrapier D 2007 Rats 1.5mg/kg MaleBrioni 1994 Rats 1.99mg/kg MaleCarro-Juárez M 2012 Rats 2mg/kg MaleCarro-Juárez M 2012 Rats 2mg/kg MalePellow S 1985 Rats 2mg/kg MaleFaturi CB 2010 Rats 2mg/kg MaleSchmitt 2002 Rats 2mg/kg MaleVolke V 1998 Rats 2mg/kg MaleMolander AC 2011 Rats 2mg/kg MaleSchmitt 2001 Rats 2mg/kg MaleContreras CM 2011 Rats 2mg/kg MaleSantos Rosa D 2012 Rats 2mg/kg MaleGriebel G 1999 Rats 2mg/kg MaleMolander AC 2011 Rats 2mg/kg MaleGriebel G 1998 Rats 2mg/kg MaleFernandez F 2004 Rats 2mg/kg MaleEngin E 2009 Rats 2mg/kg MalePain L 1999 Rats 2mg/kg MalePaine TA 2002 Rats 2mg/kg MaleKaraka A 2011 Rats 2mg/kg MaleTatarczy ska E 2004 Rats 2.5mg/kg MaleWeso owska A 2007 Rats 2.5mg/kg MaleKlodzinska A 2004 Rats 2.5mg/kg MaleKlodzinska 2004 Rats 2.5mg/kg MaleDunn RW 1989 Rats 2.5mg/kg Malede Castro PC 2007 Rats 2.5mg/kg MaleJastrz bska-Wi sek M 2014 Rats 2.5mg/kg MaleDrapier D 2007 Rats 2.5mg/kg MaleYasumatsu H 1994 Rats 2.5mg/kg MaleFlores JA 2006 Rats 2.5mg/kg MaleSimpson J 2012 Rats 2.5mg/kg MalePopik P 2006 Rats 2.5mg/kg MaleWada T 1991 Rats 2.5mg/kg MaleBrioni 1994 Rats 2.99mg/kg MaleDunn 1998 Rats 3mg/kg MaleStemmelin J 2008 Rats 3mg/kg MaleGriebel G 2002 Rats 3mg/kg MaleGriebel G 1999 Rats 3mg/kg MaleSteiner MA 2012 Rats 3mg/kg MaleOkuyama S 1999 Rats 3mg/kg MaleHarada K 2006 Rats 3.2mg/kg MaleCarro-Juárez M 2012 Rats 4mg/kg MaleMoreira MR 2014 Rats 5mg/kg MaleTatarczy ska E 2004 Rats 5mg/kg MaleKlodzinska A 2004 Rats 5mg/kg MaleKlodzinska 2004 Rats 5mg/kg MaleWeso owska A 2007 Rats 5mg/kg MaleYasumatsu H 1994 Rats 5mg/kg MaleJastrz bska-Wi sek M 2014 Rats 5mg/kg MaleHarro J 1990 Rats 5mg/kg MaleWada T 1991 Rats 5mg/kg MaleSimpson J 2012 Rats 5mg/kg MalePopik P 2006 Rats 5mg/kg MaleLangen B 2005 Rats 6mg/kg MaleHazim AI 2014 Rats 10mg/kg MaleHazim AI 2014 Rats 10mg/kg MaleOkuyama S 1999 Rats 10mg/kg MaleGriebel G 2002 Rats 10mg/kg MaleWada T 1991 Rats 10mg/kg MaleWada T 1991 Rats 10mg/kg MaleYasumatsu H 1994 Rats 10mg/kg MaleSteiner MA 2012 Rats 10mg/kg MaleHarada K 2006 Rats 10mg/kg MalePopik P 2006 Rats 10mg/kg MaleRex 2002 Rats 15mg/kg MalePopik P 2006 Rats 20mg/kg Male

NC

2684

1269

1415

6 6 6 7 6 7 7 7 7 8 5 6 51 10 8 6 7 7 7 8 5 7 12 6 18 15 8 17 7 51 10 9 18 8 8 9 6 7 7 6 8 13 12 6 6 10 10 15 9 6 6 10 10 6 6 10 12 12 9 9 10 6 5 12 8 7 13 4 8 15 10 5 24 19 18 16 17 10 9 12 7 7 13 18 10 8 6 6 6 7 7 8 6 11 8 11 15 10 8 8 10 9 12 8 10 10 10 8 10 10 10 10 10 15 18 9 17 15 12 9 8 9 8 5 8

16 11 11 20 11 6 16 8 6 20 11 10 6 11 12 8 6 5 7 9 12 10 16 15 10 10 15 13 10 5 6 10 10 10 10 20 9 9 7 11 10 11 8 9 11 8 11 11 7 10 14 12 10 12 12 8 12 7 7 6 8 8 6 7 12 11 24 11 8 7 7 7 10 12 9 8 10 10 6 10 7 11 10 10 8 8 5 6 8 7 7 8 10 8 11 20 7 6 24 12 8 6 13 11 11 12 14 16 7 8 6 7 7 8 20 8 10 24 6 24 12 8 8 14 11 12 10 20 12 16 24 10 24

Mean

25.0525.0515.68 0.6125.0517.29 1.96 1.27 0.6120.17 2.9725.05 5.2122.8020.1715.61 1.39 1.72 0.70 9.80 2.9717.29 2.3914.7217.5416.42 3.27 9.60 1.39 5.21 7.5848.4222.5020.1711.60 9.2914.22 1.96 2.2925.0516.8421.6323.7810.2714.2715.46 7.42 2.4022.3325.6625.6612.05 7.6011.5026.9315.7233.2936.9027.6916.43 8.02 7.90 4.0336.2914.6717.2938.4431.44 3.2716.42 1.0727.4714.2712.0017.5444.32 9.6035.00 4.9413.20 0.98 1.63 8.5022.50 9.8520.1725.0517.19 2.3340.8638.9016.8413.0524.85 2.0012.7816.4222.35 3.2713.6816.0535.8213.2015.9712.97 0.1712.76 5.99 1.99 2.53 3.99 4.94 5.07 0.2217.54 4.94 9.60 0.24 6.4817.0820.1732.3720.1712.6920.17

9.1115.2014.65 6.0715.20 8.21 9.11 7.00 4.07 6.0729.4518.1821.5514.6539.61 7.0013.0627.3811.4924.2711.6222.48 9.1119.2912.9613.4621.3823.87 8.6628.72 8.2110.1523.22 9.76 0.87 6.0716.2520.3818.8823.68 0.8715.2036.7419.5951.2811.6814.6512.40 4.0818.1812.1039.6122.6322.73 8.9916.4019.3310.75 3.6110.9010.9026.2152.4523.2012.0315.03 5.4229.45 7.0011.4914.9418.88 8.55 5.5312.8837.43 3.6224.1816.1818.1842.0814.6531.9418.30 0.1636.74 0.1710.9010.9010.75 3.6116.4041.0026.2129.45 6.0736.6052.4527.81 6.78 7.00 8.21 1.4512.4015.2022.7312.10 9.1111.4915.9310.9010.75 3.6110.90 6.0726.2131.30 6.7852.4527.8113.09 7.8814.3012.1012.40 6.78 3.75 6.0722.73 9.1127.81 8.4327.81

ControlSD

9.35 9.35 2.40 0.11 9.35 1.61 0.22 0.54 0.11 6.22 1.06 9.3511.10 4.32 6.22 3.73 0.22 0.21 0.11 3.58 1.06 1.61 1.4116.49 9.5026.01 3.0813.61 0.2211.10 5.1211.5112.37 6.22 6.79 3.18 6.83 0.22 0.32 9.35 5.38 8.3110.92 5.23 7.89 9.78 5.87 2.26 8.69 6.70 6.70 3.54 5.48 3.92 7.32 3.3710.0510.2914.7010.00 7.49 2.94 2.9114.0618.05 1.6111.47 7.24 3.0826.01 1.3730.3215.3512.64 9.50 8.0613.6117.71 2.9110.45 0.11 0.22 7.5712.3710.90 6.22 9.35 2.28 0.42 5.62 3.12 5.38 5.59 2.89 1.92 3.9826.01 6.97 3.0816.6810.9710.0110.4524.55 8.75 0.13 8.43 5.13 1.57 1.60 2.89 3.50 3.47 0.09 9.50 2.9113.61 0.10 7.9211.25 6.2216.19 6.22 3.15 6.22

4.2216.9118.51 3.7416.91 4.01 4.22 3.49 1.31 3.7411.2115.25 4.4318.5127.44 3.4912.7114.46 5.24 8.80 6.4011.44 4.2214.94 6.63 5.7913.0518.15 7.7520.34 4.01 8.03 6.86 7.13 1.10 3.7424.8013.51 4.3711.38 1.1016.9112.2310.3214.91 4.5318.51 6.63 2.6215.25 5.5427.44 4.99 9.00 5.74 5.09 7.42 4.27 1.37 3.43 3.9621.8517.5914.1915.9515.55 6.5311.21 3.49 5.2410.44 4.3710.72 4.7010.8211.24 5.0119.41 8.4715.2518.3818.5123.2315.83 0.1312.23 0.05 3.43 3.96 4.27 1.37 5.09 8.4321.8511.21 3.7413.2317.5937.68 5.94 3.49 4.01 2.65 6.6316.91 9.00 5.54 4.22 5.24 3.36 3.43 4.27 1.37 3.96 3.7421.8527.51 8.4017.5937.6828.02 7.80 8.93 5.54 6.63 5.94 5.56 3.74 9.00 4.2237.68 5.3837.68

NT

2557

1172

1385

6 6 6 7 6 7 7 7 7 8 7 6 21 10 7 6 7 7 7 8 6 7 12 6 18 18 8 17 7 22 10 9 8 8 8 9 6 7 7 6 8 13 12 6 6 10 10 15 9 6 6 10 10 6 6 10 12 12 8 9 10 6 5 12 8 7 13 4 8 19 10 5 10 19 17 16 17 10 9 12 7 7 13 8 10 7 6 6 6 7 7 8 6 11 8 11 20 10 8 8 10 9 12 8 10 10 10 8 10 10 10 10 10 15 20 9 17 15 8 9 4 9 4 5 3

16 11 11 20 11 6 16 6 6 20 12 10 6 11 12 8 6 6 7 9 7 10 16 15 10 10 10 13 10 5 6 10 10 10 10 20 10 9 7 11 10 11 8 9 11 10 11 11 7 10 14 12 10 12 12 8 12 7 7 7 8 8 6 7 12 11 8 11 8 7 7 7 10 9 9 8 8 6 6 10 7 11 10 10 8 8 5 6 8 7 7 8 10 8 11 20 7 6 24 12 8 6 13 11 11 12 14 16 7 8 7 7 7 8 20 8 10 24 6 24 12 8 8 14 11 12 10 20 12 16 24 10 24

Mean

36.7231.7231.84 0.9440.8915.46 1.27 0.61 0.2823.4314.8355.8920.9531.4729.3346.17 3.72 4.38 1.0620.0210.0330.36 5.2841.2230.2236.94 5.0112.60 1.0616.4540.9968.8020.0036.5324.4032.3266.83 9.6313.2769.9438.3965.8561.4135.6737.7350.0847.01 8.4251.9957.3157.3127.0518.8721.7057.2445.2759.5859.7565.7940.7120.3815.3012.6359.0246.9232.3164.5544.6711.1150.75 7.3766.0533.2723.8029.8562.7316.0058.33 8.2814.96 0.98 1.6323.6039.1719.2143.5381.8955.85 8.2469.4968.9555.4870.9837.5511.1736.2765.3040.9110.2450.0744.9657.4736.8150.7042.67 0.3732.3822.8629.5529.6029.5329.8129.65 0.3130.6010.3525.60 0.3112.7636.6759.3372.8444.0045.9733.77

11.8820.1025.81 8.4017.90 7.4611.7513.50 5.7711.9243.3427.1321.8714.3036.8417.0389.2993.7329.8860.7562.8553.2922.1858.3835.3735.1761.3061.3028.9169.0223.9822.1143.1321.4416.6210.7749.0438.1324.1439.4616.2833.2044.0533.8869.8724.1623.7216.50 4.9623.0013.7043.7124.7423.0515.8624.1325.6820.70 6.7220.6020.6043.3152.7754.5165.1155.4519.5847.2321.6746.7652.7629.9747.0343.3635.0256.4640.4658.1947.8938.0262.7143.2656.3744.16 0.4240.75 0.1847.2047.2047.3115.7133.7073.0057.3857.7812.6252.2069.4255.62 7.7314.9724.5411.9532.9047.8035.0619.8829.8319.6233.1570.4070.4323.4670.4015.4859.5974.7016.2372.7164.5028.3846.6138.5140.0049.1019.6621.2112.2732.1428.5158.8818.5777.22

TreatmentSD

6.1514.18 9.97 0.2210.66 4.54 1.73 0.11 0.11 8.58 7.5812.7815.62 8.64 9.43 4.25 0.43 0.54 0.11 4.47 4.6015.48 3.3341.3720.5828.90 3.7010.31 0.1327.9122.28 8.8614.14 8.3040.80 3.64 8.74 1.73 2.48 8.18 5.3814.8911.11 9.80 7.6713.7519.50 2.2613.8115.8015.80 7.79 3.79 4.4115.9617.6913.3110.2920.2113.93 4.76 4.41 5.4410.4717.2711.7318.38 7.98 6.4723.12 6.1131.6120.5817.4420.5846.05 9.4860.6621.0013.06 0.11 0.3311.9015.91 9.3514.61 2.26 7.19 1.24 3.15 6.31 7.8416.02 2.89 4.1613.0215.90 9.05 3.0816.1818.2913.8416.2913.7524.45 0.1317.6216.9030.6330.6630.5230.4430.71 0.0819.00 8.3624.33 0.1032.6633.7544.7816.1947.5212.4156.10

7.3923.5515.62 5.1125.87 7.86 5.8127.48 0.33 6.8818.4319.17 7.8911.5716.9229.6011.72 8.52 4.1913.2027.9413.40 7.9222.9113.9414.5628.9326.9217.0128.8413.63 9.7320.5810.6018.07 4.3330.5817.44 5.4819.3521.9025.21 7.2828.5941.0731.4825.4515.92 2.4414.81 7.85 6.8659.6512.37 3.45 5.75 5.1415.65 4.9215.1917.5428.0913.8710.2334.9830.0324.4925.8028.53 7.8616.35 5.9325.6433.6114.9412.0333.9526.4428.3413.5012.6626.0320.0334.77 0.7814.26 0.0311.0212.7313.51 5.20 7.1724.2424.9729.48 7.8718.5217.2349.28 8.9724.0011.29 8.9020.8931.5114.49 9.7013.73 9.44 4.2026.7028.4510.1230.83 7.2822.6350.28 9.8923.6553.6350.9414.85 8.9318.0223.8810.4917.40 8.0613.5034.3252.1816.3462.32

-10 -5 0 5 10

Anxiolytic Anxiogenic

Standardised mean difference SMD

-1.34

-1.60

-1.14

-1.36-0.51-2.06-1.79-1.46 0.50 0.53 1.57 2.83-0.41-1.85-2.54-1.24-1.21-1.10-7.05-6.38-6.06-3.18-2.39-1.84-1.11-1.09-0.78-0.77-0.72-0.48-0.24 1.73-0.62-1.98-1.89 0.19-2.11-0.41-6.42-6.19-5.83-5.80-4.72-3.79-3.55-3.30-2.98-2.78-2.78-2.63-2.58-2.45-2.41-2.41-2.38-2.29-2.26-2.25-2.22-2.15-2.14-2.07-1.91-1.89-1.82-1.78-1.77-1.73-1.68-1.65-1.51-1.46-1.37-1.36-1.12-1.09-0.76-0.76-0.54-0.53-0.50-0.21-0.14 0.00 0.00-1.47-1.20-0.88-2.01-7.71-6.69-5.89-5.89-5.65-5.43-4.46-4.23-2.68-2.35-2.30-2.20-2.14-2.09-1.84-1.71-1.67-1.65-1.55-1.48-1.36-1.28-1.22-1.19-1.13-1.10-1.08-1.05-0.84-0.82-0.79-0.69-0.28-0.74-1.44-2.38-0.83-3.32-0.46

-0.45-0.23-0.63-0.51-0.12 0.11-0.51-0.34-1.65-1.04-0.87-0.50-0.05 0.02 0.12-0.45-5.75-5.25-3.63-3.10-2.82-2.37-2.01-1.97-1.97-1.88-1.86-1.58-1.47-1.46-1.45-1.28-1.24-1.24-1.18-1.14-1.12-1.08-0.99-0.96-0.95-0.81-0.69-0.63-0.58-0.50-0.39-0.32-0.32-0.31-0.23-0.20-0.05-0.03-1.40-1.35-0.96-0.81-0.80-0.79-0.72-0.64-0.02-2.37-1.88-1.63-1.05-0.86-0.68-4.94-2.58-1.99-1.87-1.64-1.62-1.55-1.54-1.45-1.40-1.32-1.22-1.22-1.08-0.92-0.44-0.29-0.28-4.10-3.64-3.41-2.98-2.63-1.69-1.26-1.22-1.04-0.91-0.90-0.62-0.12-0.44-1.78-1.55-1.27-1.24-0.99-0.96-1.99-1.00-4.28-2.79-2.75-2.57-2.56-1.59-1.42-1.03-1.01-0.90-0.78-0.36-3.09-2.56-2.03-2.01-1.46-1.29-0.97-0.79-0.77-0.67-0.80-0.94

95%-CI

[ -1.46; -1.227]

[ -1.80; -1.398]

[ -1.26; -1.009]

[ -2.67; -0.053][ -1.67; 0.646][ -3.56; -0.552][ -3.09; -0.484][ -2.79; -0.126][ -0.57; 1.572][ -0.54; 1.600][ 0.32; 2.818][ 1.22; 4.440][ -1.41; 0.581][ -3.31; -0.399][ -4.21; -0.874][ -1.79; -0.691][ -2.19; -0.244][ -2.21; 0.014][-10.64; -3.472][ -9.33; -3.426][ -8.88; -3.236][ -4.91; -1.450][ -3.75; -1.026][ -3.36; -0.325][ -2.26; 0.041][ -1.96; -0.223][ -1.97; 0.415][ -1.45; -0.094][ -1.43; -0.015][ -1.48; 0.514][ -0.92; 0.432][ 0.44; 3.016][ -1.14; -0.114][ -3.09; -0.870][ -3.04; -0.734][ -0.65; 1.022][ -3.40; -0.822][ -1.41; 0.580][ -8.95; -3.889][ -9.37; -2.998][ -8.57; -3.102][ -8.52; -3.083][ -7.25; -2.177][ -5.58; -1.996][ -4.85; -2.255][ -4.59; -2.006][ -4.81; -1.155][ -4.54; -1.029][ -4.07; -1.482][ -3.89; -1.373][ -3.59; -1.584][ -3.74; -1.159][ -4.03; -0.787][ -4.03; -0.787][ -3.57; -1.178][ -3.47; -1.111][ -3.83; -0.687][ -3.82; -0.685][ -3.39; -1.060][ -3.19; -1.114][ -3.18; -1.107][ -3.30; -0.832][ -3.07; -0.748][ -2.98; -0.796][ -3.25; -0.390][ -3.36; -0.197][ -2.74; -0.802][ -2.92; -0.531][ -2.95; -0.404][ -2.56; -0.741][ -3.22; 0.205][ -2.60; -0.325][ -2.13; -0.612][ -2.36; -0.368][ -2.51; 0.262][ -1.88; -0.305][ -1.42; -0.098][ -1.45; -0.069][ -1.25; 0.164][ -1.22; 0.153][ -1.39; 0.393][ -1.14; 0.715][ -0.95; 0.657][ -1.05; 1.048][ -1.05; 1.048][ -2.35; -0.585][ -2.10; -0.292][ -1.81; 0.045][ -3.32; -0.701][-11.60; -3.827][-10.11; -3.275][ -8.94; -2.834][ -8.64; -3.133][ -8.31; -2.993][ -7.80; -3.070][ -6.88; -2.029][ -5.84; -2.626][ -4.12; -1.233][ -3.48; -1.217][ -3.18; -1.418][ -3.36; -1.044][ -3.43; -0.844][ -3.38; -0.810][ -2.92; -0.756][ -2.82; -0.591][ -2.62; -0.716][ -2.83; -0.472][ -2.57; -0.523][ -2.50; -0.470][ -2.35; -0.367][ -2.38; -0.174][ -2.19; -0.246][ -2.16; -0.226][ -2.09; -0.170][ -2.05; -0.145][ -2.03; -0.125][ -1.82; -0.280][ -1.50; -0.171][ -1.80; 0.149][ -1.49; -0.091][ -1.43; 0.047][ -1.18; 0.617][ -1.70; 0.222][ -2.83; -0.055][ -3.65; -1.109][ -2.09; 0.436][ -5.53; -1.104][ -1.81; 0.888]

[ -1.15; 0.254][ -1.07; 0.609][ -1.49; 0.233][ -1.14; 0.121][ -0.96; 0.718][ -1.02; 1.243][ -1.21; 0.199][ -1.41; 0.729][ -3.03; -0.265][ -1.70; -0.371][ -1.73; -0.005][ -1.39; 0.398][ -1.18; 1.084][ -0.81; 0.858][ -0.68; 0.918][ -1.45; 0.546][ -8.75; -2.759][ -8.20; -2.305][ -5.51; -1.740][ -4.56; -1.632][ -4.18; -1.450][ -3.56; -1.172][ -2.88; -1.139][ -2.86; -1.075][ -3.07; -0.859][ -2.96; -0.788][ -2.84; -0.883][ -2.48; -0.681][ -2.48; -0.456][ -2.94; 0.021][ -2.78; -0.119][ -2.27; -0.303][ -2.22; -0.268][ -2.21; -0.264][ -2.14; -0.213][ -1.81; -0.468][ -2.10; -0.134][ -2.09; -0.077][ -2.13; 0.138][ -1.85; -0.065][ -1.89; -0.016][ -1.68; 0.069][ -1.70; 0.330][ -1.59; 0.319][ -1.44; 0.278][ -1.45; 0.448][ -1.24; 0.453][ -1.17; 0.519][ -1.38; 0.734][ -1.19; 0.576][ -0.97; 0.514][ -1.00; 0.605][ -0.92; 0.829][ -0.83; 0.771][ -2.31; -0.492][ -2.46; -0.231][ -1.81; -0.108][ -1.92; 0.293][ -1.91; 0.300][ -1.94; 0.360][ -1.74; 0.299][ -1.65; 0.370][ -1.15; 1.113][ -3.83; -0.904][ -2.87; -0.896][ -2.61; -0.638][ -1.90; -0.205][ -1.74; 0.021][ -1.70; 0.334][ -7.32; -2.560][ -4.11; -1.050][ -3.35; -0.636][ -2.96; -0.787][ -2.67; -0.619][ -2.72; -0.519][ -2.70; -0.390][ -2.63; -0.452][ -2.61; -0.286][ -2.72; -0.082][ -2.31; -0.332][ -2.40; -0.050][ -2.14; -0.294][ -2.03; -0.127][ -1.85; 0.015][ -1.43; 0.557][ -1.27; 0.701][ -1.53; 0.971][ -6.38; -1.825][ -5.38; -1.898][ -5.23; -1.604][ -4.64; -1.319][ -4.06; -1.201][ -2.74; -0.637][ -2.35; -0.157][ -2.15; -0.297][ -1.71; -0.378][ -2.03; 0.211][ -2.11; 0.313][ -1.20; -0.043][ -0.92; 0.680][ -1.43; 0.556][ -3.20; -0.360][ -2.44; -0.655][ -2.20; -0.340][ -2.17; -0.313][ -1.84; -0.131][ -1.74; -0.168][ -2.85; -1.123][ -2.13; 0.137][ -6.24; -2.324][ -4.46; -1.114][ -4.33; -1.162][ -4.10; -1.044][ -3.97; -1.150][ -2.31; -0.874][ -2.55; -0.289][ -1.97; -0.081][ -1.62; -0.409][ -2.11; 0.315][ -1.37; -0.190][ -1.17; 0.449][ -4.65; -1.520][ -3.97; -1.152][ -2.97; -1.096][ -3.08; -0.954][ -2.38; -0.541][ -2.28; -0.312][ -1.63; -0.309][ -1.63; 0.044][ -1.50; -0.052][ -1.25; -0.089][ -1.72; 0.121][ -1.54; -0.345]

W(random)

100%

45.2%

54.8%

0.3% 0.4% 0.3% 0.3% 0.3% 0.4% 0.4% 0.4% 0.3% 0.4% 0.3% 0.3% 0.6% 0.4% 0.4% 0.1% 0.1% 0.1% 0.3% 0.3% 0.3% 0.4% 0.5% 0.4% 0.5% 0.5% 0.4% 0.5% 0.4% 0.6% 0.4% 0.4% 0.5% 0.4% 0.4% 0.2% 0.1% 0.1% 0.1% 0.2% 0.2% 0.3% 0.4% 0.2% 0.3% 0.3% 0.4% 0.4% 0.4% 0.3% 0.3% 0.4% 0.4% 0.3% 0.3% 0.4% 0.4% 0.4% 0.4% 0.4% 0.4% 0.3% 0.3% 0.4% 0.4% 0.4% 0.5% 0.3% 0.4% 0.5% 0.4% 0.3% 0.5% 0.5% 0.5% 0.5% 0.5% 0.5% 0.4% 0.5% 0.4% 0.4% 0.5% 0.5% 0.4% 0.3% 0.1% 0.1% 0.1% 0.1% 0.1% 0.2% 0.2% 0.3% 0.3% 0.4% 0.5% 0.4% 0.3% 0.4% 0.4% 0.4% 0.4% 0.4% 0.4% 0.4% 0.4% 0.4% 0.4% 0.4% 0.4% 0.4% 0.4% 0.5% 0.5% 0.4% 0.5% 0.5% 0.5% 0.4% 0.3% 0.4% 0.4% 0.2% 0.3%

0.5% 0.5% 0.5% 0.5% 0.5% 0.4% 0.5% 0.4% 0.3% 0.5% 0.5% 0.5% 0.4% 0.5% 0.5% 0.4% 0.1% 0.1% 0.2% 0.3% 0.3% 0.4% 0.5% 0.5% 0.4% 0.4% 0.4% 0.5% 0.4% 0.3% 0.3% 0.4% 0.4% 0.4% 0.4% 0.5% 0.4% 0.4% 0.4% 0.5% 0.4% 0.5% 0.4% 0.4% 0.5% 0.4% 0.5% 0.5% 0.4% 0.5% 0.5% 0.5% 0.5% 0.5% 0.5% 0.4% 0.5% 0.4% 0.4% 0.4% 0.4% 0.4% 0.4% 0.3% 0.4% 0.4% 0.5% 0.5% 0.4% 0.2% 0.3% 0.3% 0.4% 0.4% 0.4% 0.4% 0.4% 0.4% 0.3% 0.4% 0.4% 0.4% 0.4% 0.4% 0.4% 0.4% 0.4% 0.2% 0.3% 0.2% 0.3% 0.3% 0.4% 0.4% 0.4% 0.5% 0.4% 0.4% 0.5% 0.5% 0.4% 0.3% 0.5% 0.4% 0.4% 0.5% 0.5% 0.5% 0.4% 0.2% 0.3% 0.3% 0.3% 0.3% 0.5% 0.4% 0.4% 0.5% 0.4% 0.5% 0.5% 0.3% 0.3% 0.4% 0.4% 0.4% 0.4% 0.5% 0.5% 0.5% 0.5% 0.4% 0.5%

Figure 3. Meta-analysis of diazepam on rodent anxiety related behavior.Meta-analysis of rodent diazepam effect sizes, shown as a forest plot of standardized effect sizes (Hedges’ g). The meta-analysis is sub-grouped by animal species. Error bars indicate the 95% confidence intervals of standardized mean difference (SMD). The weighted average mean effect size of subgroups and all studies is represented by the central vertices of a red diamond; the outer vertices indicate the 95% confidence intervals. Control and treatment samples sizes are given in the columns listed as NC and NT respectively.


https://doi.org/10.1101/020701


Ht1a Knockout

Random effects model

Heterogeneity: I^2=28%, Tau^2=0.1188, p=0.12

Assay = EPM

Assay = LD

Assay = OF




Heterogeneity: I^2=29.7%, Tau^2=0.1777, p=0.1813


Heterogeneity: I^2=0%, Tau^2=0, p=0.4802

Groenink, 2003 Mice Female and MaleGross, 2002 Mice Female and MalePiszczek, 2013 Mice Female and MaleRamboz, 1998 Mice Female and MaleFerres Coy, 2013 Mice MaleFreeman Daniels, 2011 Mice MaleGleason, 2010 Mice MaleGleason, 2010 Mice MaleGleason, 2010 Mice Male

Groenink, 2003 Mice Female and MaleKlemenhagen, 2006 Mice Male

Ramboz, 1998 Mice FemaleGroenink, 2003 Mice Female and MaleGross, 2002 Mice Female and MalePiszczek, 2013 Mice Female and MaleGleason, 2010 Mice MaleJain, 2012 Mice MaleParks, 1998 Mice MaleRamboz, 1998 Mice MaleVinkers, 2010 Mice Male

N

324

153

22

149

8 16 27 20 12 43 9 11 7

12 10

20 16 16 28 9 14 15 20 11

Mean

4.1046.3919.6728.5329.4821.3016.6122.5329.35

24.1643.69

0.83 9.69 4.7214.75 8.2211.07 5.43 3.0210.09

KO

SD

3.1114.7012.6618.3112.3615.15 8.4710.7212.37

29.3433.43

0.9611.66 3.66 8.81 2.84 7.38 5.05 4.56 6.86

N

335

152

28

155

8 16 30 20 12 39 9 11 7

12 16

20 16 16 34 9 14 15 20 11

Mean

14.3930.2011.5417.1714.9313.23 5.9710.3116.96

35.9118.20

0.47 6.52 1.20 8.93 3.34 4.83 1.41 1.03 1.29

WT

SD

16.6826.62 5.6313.9312.0313.2210.8910.1411.79

37.3618.09

0.72 4.46 2.15 4.23 3.93 4.33 1.81 2.88 6.00

2 1 0 1 2 3



0.73

0.71

0.32

0.80

0.81 0.73 0.83 0.68 1.15 0.56 1.04 1.13 0.96

0.34 0.99

0.41 0.35 1.14 0.86 1.35 1.00 1.03 0.51 1.31

95% CI

[ 0.50; 0.96]

[ 0.33; 1.09]

[ 0.98; 1.62]

[ 0.56; 1.04]

[ 1.84; 0.22][ 0.02; 1.45][ 0.29; 1.38][ 0.05; 1.32][ 0.28; 2.03][ 0.12; 1.00][ 0.04; 2.04][ 0.22; 2.04][ 0.17; 2.09]

[ 1.14; 0.47][ 0.15; 1.83]

[ 0.22; 1.04][ 0.35; 1.05][ 0.39; 1.90][ 0.34; 1.38][ 0.30; 2.40][ 0.21; 1.79][ 0.26; 1.80][ 0.12; 1.14][ 0.37; 2.25]

W(random)

100%

44.3%

9.1%

46.6%

3.4% 5.3% 6.9% 6.0% 4.2% 7.9% 3.6% 4.0% 3.0%

4.7% 4.4%

6.1% 5.5% 5.0% 7.1% 3.3% 4.8% 4.9% 6.1% 3.9%

Figure 4. Meta-analyses of serotonin receptor interventions on rodent anxiety-related behaviors.

Meta-analysis of effect sizes of serotonin-targeted interventions is shown as a forest plot of standardized effect sizes (Hedges’ g). Error bars indicate the 95% confidence intervals of g. The weighted average meaneffect size of all studies is represented by the central vertices of a red diamond; the outer vertices indicate the 95% confidence intervals. Control and treatment samples sizes (N), the assay types of the studies,and the assay types of the studies are given; elevated plus maze (EPM), open field (OF) and light-dark box (LD). Effects of: A. Serotonin receptor gene (Htr1a) knockout models B. Htr1a overexpression models.

A

B Ht1a Over-expression



Assay = EPM

Assay = OF





Kusserow, 2004 Mouse Female EPMAudero, 2013 Mouse Male EPMBert, 2006 Mouse Male EPMBert, 2006 Mouse Male EPMKusserow, 2004 Mouse Male EPM

Kusserow, 2004 Mouse Female OFAudero, 2013 Mouse Male OFKusserow, 2004 Mouse Male OF

N

100

62

38

13 12 11 13 13

13 12 13

Mean

6.9529.2814.93 9.08 6.69

11.6433.2811.94

OE

SD

6.20 7.42 8.58 5.96 6.99

34.75 8.2529.87

N

98

63

35

1215111312

121112

Mean

18.6429.3717.2927.3416.48

9.7028.81 9.29

WT

SD

4.6517.15 8.9610.59 6.53

28.17 6.0828.17

-2 0 2 4



-0.599

-1.117

0.231

-2.049-0.006-0.258-2.058-1.398

0.059 0.590 0.088

95%-CI

[-1.32; 0.13]

[-1.97; -0.26]

[-0.23; 0.69]

[-3.05; -1.05][-0.77; 0.75][-1.10; 0.58][-3.04; -1.08][-2.29; -0.51]

[-0.73; 0.84][-0.25; 1.43][-0.70; 0.87]

W(random)

100%

61.6%

38.4%

11.8%13.0%12.6%11.9%12.3%

12.9%12.6%12.9%

-4


https://doi.org/10.1101/020701


Htt Knockout



Assay = EPM

Assay = LD

Assay = OF







Holmes, 2003 Mice FemaleHolmes, 2003 Mice FemaleLi, 2004 Mice FemaleCarroll, 2007 Mice Female and MaleLine, 2011 Mice Female and MaleHolmes, 2003 Mice MaleHolmes, 2003 Mice MaleHolmes, 2003 Mice MaleKalueff, 2007 Mice MaleKalueff, 2007 Mice MaleLira, 2003 Mice MaleSchipper, 2011 Rats FemaleOlivier, 2008 Rats Male

Holmes, 2003 Mice FemaleHolmes, 2003 Mice FemaleCarroll, 2007 Mice Female and MaleHolmes, 2003 Mice MaleHolmes, 2003 Mice MaleHolmes, 2003 Mice Male

Kalueff, 2007 Mice FemaleCarroll, 2007 Mice Female and MaleZhao, 2006 Mice Female and MaleZhao, 2006 Mice Female and MaleHolmes, 2003 Mice MaleKalueff, 2007 Mice MaleKalueff, 2007 Mice MaleMoya, 2011 Mice MalePang, 2011 Mice MaleOlivier, 2008 Rats Male

N

412

173

94

145

11 14 11 30 9 15 11 13 11 11 20 5 12

11 14 30 15 11 13

8 30 12 12 14 11 11 10 25 12

Mean

16.26 6.03 48.38 49.68 10.82 45.86 12.30 5.75292.00 48.67 6.79 38.45 10.14

23.01 5.33 47.98 29.28 19.06 3.76

42.92 20.11 36.69 34.14 39.63 64.00 42.07 55.97 54.68 29.72

KO

SD

14.21 9.98 25.83 27.69 6.62 34.16 7.61 8.51

126.03 21.01 5.92 10.52 8.25

13.93 6.51 14.17 23.68 7.51 7.47

22.95 13.52 25.05 22.92 11.88 19.90 9.04 18.96 31.75 15.89

N

388

158

90

140

13 11 11 29 7 15 10 12 8 8 17 5 12

13 11 29 15 10 12

7 29 12 12 14 8 8 10 28 12

Mean

5.16 4.96 14.87 41.51 2.94 19.76 1.40 0.04168.00 28.00 7.67 18.58 5.56

7.57 6.46 32.93 18.70 4.59 1.05

6.82 12.82 18.96 16.93 17.17 37.00 25.16 22.70 48.68 15.61

WT

SD

5.830 8.915 21.915 34.514 5.601 21.859 2.833 0.001

138.593 23.099 7.999 5.521 5.498

7.769 11.361 20.900 12.297 4.106 2.967

13.067 13.296 12.526 9.471 15.401 22.627 11.333 10.410 28.003 16.191

2 0 2 4



0.88

0.78

0.83

1.01

1.02 0.11 1.35 0.26 1.20 0.89 1.79 0.90 0.90 0.900.12

2.14 0.63

1.350.12

0.83 0.55 2.26 0.45

1.78 0.54 0.86 0.95 1.59 1.22 1.61 2.08 0.20 0.85

95% CI

[ 0.648; 1.10]

[ 0.450; 1.10]

[ 0.171; 1.49]

[ 0.658; 1.36]

[ 0.158; 1.88][ 0.682; 0.90][ 0.403; 2.29][ 0.254; 0.77][ 0.105; 2.30][ 0.131; 1.64][ 0.744; 2.83][ 0.068; 1.73][ 0.064; 1.87][ 0.064; 1.87][ 0.771; 0.52][ 0.424; 3.85][ 0.192; 1.46]

[ 0.450; 2.26][ 0.913; 0.67][ 0.301; 1.37][ 0.185; 1.28][ 1.122; 3.41][ 0.343; 1.25]

[ 0.531; 3.04][ 0.016; 1.06][ 0.021; 1.71][ 0.096; 1.80][ 0.721; 2.45][ 0.215; 2.23][ 0.533; 2.68][ 0.952; 3.22][ 0.343; 0.74][ 0.008; 1.69]

W(random)

100%

43.7%

22.1%

34.2%

3.4% 3.7% 3.1% 5.0% 2.6% 3.9% 2.8% 3.6% 3.1% 3.1% 4.4% 1.4% 3.6%

3.3% 3.7% 4.9% 4.0% 2.5% 3.7%

2.2% 5.0% 3.5% 3.5% 3.4% 2.9% 2.7% 2.5% 4.9% 3.5%

Figure 5. Meta-analyses of serotonin transporter interventions on rodent anxiety-related behaviors.

Meta-analysis of effect sizes of serotonin-targeted interventions is shown as a forest plot of standardized effect sizes (Hedges’ g). Error bars indicate the 95% confidence intervals of g. The weighted average meaneffect size of all studies is represented by the central vertices of a red diamond; the outer vertices indicate the 95% confidence intervals. Control and treatment samples sizes (N), and the assay types of the studies are given; elevated plus maze (EPM), open field (OF) and light-dark box (LD). Effects of: A. Serotonin transporter gene (Htt) knockout models B. Htt overexpression models.

A

BHtt Overexpression



Jennings, 2006 Mouse Male EPMJennings, 2006 Mouse Male EPMLine, 2011 Mouse Male and Female EPMJennings, 2006 Mouse Male OF

N

33

8 8 9 8

Mean

15.52 1.5918.3438.69

OE

SD

14.63 2.7414.2929.27

N

31

8 8 7 8

Mean

30.6928.8439.1636.34

WT

SD

12.3121.7015.6321.95

2 1 0 1 2 3


Standardised mean difference

SMD

0.94

1.061.671.32

0.09

95% CI

[ 1.69; 0.202]

[ 2.13; 0.005][ 2.85; 0.484][ 2.44; 0.205][ 0.89; 1.067]

W(random)

100%

25.4%22.7%24.2%27.7%


https://doi.org/10.1101/020701


Figure 6. Meta-analyses of the effects of stress signaling genes on anxiety-related behaviors.

Meta-analysis of effect sizes of stress signaling genes, shown as a forest plot of standardized effect sizes (Hedges’ g). Error bars indicate the 95% confidence intervals of g. The weighted average meaneffect size of all studies is represented by the central vertices of a red diamond; the outer vertices indi-cate the 95% confidence intervals. Control and treatment samples sizes (N), and the assay typesof the studies are given; elevated plus maze (EPM), open field (OF) and light-dark box (LD). Effects of: A. Crh gene knockout. B. Crhr1 gene knockout.

A

B

Crhr1 Knockout


Heterogeneity: I^2=13.1%, Tau^2=0, p=0.3278

Gammie, 2006 Mouse Male EPMMuller, 2003 Mouse Male EPMSmith, 1998 Mouse Male EPMTrimble, 2007 Mouse Male and Female EPMLiebsch, 1995 Rats Male EPMLiebsch, 1999 Rats Male EPMMuller, 2003 Mouse Male LDSmith, 1998 Mouse Male LD

N

105

19 20 9 14 10 7 20 6

Mean

35.9812.3516.5815.6413.8514.29 3.97 9.88

KO

SD

31.2722.0916.9319.36 6.1315.40 6.3120.74

N

99

2120 8 611 720 6

Mean

62.9125.4071.9759.0345.0742.3315.8856.80

WT

SD

40.9531.5651.8834.5622.5221.9313.8163.08

2 1 0 1 2 3



SMD

1.00

0.720.471.401.691.781.391.090.92

95% CI

[ 1.30; 0.70]

[ 1.36; 0.08][ 1.10; 0.16][ 2.49; 0.31][ 2.81; 0.57][ 2.82; 0.73][ 2.59; 0.18][ 1.76; 0.42][ 2.14; 0.29]

W(random)

100%

21.8%22.7% 7.6% 7.2% 8.3% 6.2%20.1% 6.1%

Crh Knockout



Dunn, 1999 Mouse Male EPMWeninger, 1999 Mouse Male EPM

N

20

812

Mean

45.7447.86

KO

SD

17.9630.14

N

21

912

Mean

40.6939.39

WT

SD

17.9919.15

1 0 1 2



SMD

0.30

0.270.32

95% CI

[ 0.32; 0.92]

[ 0.69; 1.22][ 0.48; 1.13]

W(random)

100%

41.5%58.5%


https://doi.org/10.1101/020701


Physical Restraint



Assay = EPM

Assay = LD

Assay = OF







Harris, 2001 12 mins Mice MaleLunga, 2004 60 mins Rats FemaleCarvajal, 2004 60 mins Rats Female and MaleRylkova, 2009 15 mins Rats MaleWalf, 2012 20 mins Rats MaleLiu, 2013 30 mins Rats MaleLocchi, 2007 45 mins Rats MaleAnand, 2012 60 mins Rats MaleBusnardo, 2013 60 mins Rats MaleEstanislau, 2005 60 mins Rats MaleGranjeiro, 2011 60 mins Rats MaleJoshi JC, 2014 60 mins Rats MaleNosek, 2008 60 mins Rats MaleNosek, 2008 60 mins Rats MaleReis, 2011 60 mins Rats Male

Ouagazzal, 2003 5 mins Mice FemaleHarris, 2001 12 mins Mice Male

Chesworth, 2012 15 mins Mice MaleChesworth, 2012 15 mins Mice MaleNosek, 2008 60 mins Rats MaleNosek, 2008 60 mins Rats Male

N

214

143

29

42

14 7 12 9 24 9 9 6 10 11 6 6 7 7 6

15 14

11 17 7 7

Mean

15.6728.7223.9228.93 8.4741.8615.4419.3014.2429.3711.4311.88 9.66 6.8756.56

4.98 1.67

25.7126.70 3.44 5.35

Unrestrained

SD

9.35 6.29 3.5925.40 9.8025.40 5.48 6.86 6.9913.16 5.65 4.57 6.45 4.1632.55

7.68 0.62

9.6710.45 1.85 1.86

N

214

143

29

42

14 7 12 9 24 10 9 6 7 12 7 6 7 7 6

15 14

11 17 7 7

Mean

25.6713.78 8.9531.87 4.40 9.53 5.01 4.10 6.1313.05 5.11 1.54 6.8716.3719.05

4.47 2.50

19.3429.1829.3710.70

Restrained

SD

6.24 6.71 3.2831.75 7.3512.64 2.17 2.94 5.0316.50 5.74 2.14 5.00 6.8315.84

4.65 1.25

9.1911.6823.26 5.97

4 2 0 2 4 6


Standardised mean differenceSMD

0.66

1.14

0.36

0.46

1.22 2.15 4.200.10

0.46 1.57 2.38 2.66 1.22 1.05 1.03 2.67 0.451.57

1.35

0.080.82

0.650.221.471.13

95% CI

[ 0.005; 1.32]

[ 0.376; 1.90]

[ 1.239; 0.52]

[ 1.392; 0.47]

[ 2.038; 0.40][ 0.749; 3.55][ 2.680; 5.72][ 1.022; 0.83][ 0.112; 1.04][ 0.509; 2.63][ 1.110; 3.65][ 0.949; 4.37][ 0.152; 2.30][ 0.166; 1.93][ 0.155; 2.22][ 0.959; 4.39][ 0.613; 1.52][ 2.821; 0.32][ 0.047; 2.66]

[ 0.637; 0.80][ 1.596; 0.04]

[ 0.213; 1.51][ 0.893; 0.46][ 2.696; 0.25][ 2.290; 0.02]

W(random)

100%

70.0%

10.3%

19.6%

5.1% 4.4% 4.3% 5.0% 5.3% 4.8% 4.6% 4.0% 4.8% 5.0% 4.7% 4.0% 4.8% 4.6% 4.5%

5.2% 5.1%

5.0% 5.2% 4.6% 4.7%

Social Isolation



Assay = EPM

Assay = LD

Assay = OF







Abramov, 2004 21 days Mice FemaleBlednov YA, 2001 6 days Mice Female and MaleMajercsik, 2003 7 days Mice MaleMajercsik, 2003 7 days Mice MaleMajercsik, 2003 7 days Mice MaleMajercsik, 2003 7 days Mice MaleRodgers, 1993 7 days Mice MaleEstelles J, 2007 14 days Mice MaleRodgers, 1993 14 days Mice MaleAbramov, 2004 21 days Mice MaleRodgers, 1993 21 days Mice MaleWei XY, 2007 21 days Mice MaleKoike H, 2009 28 days Mice MaleKoike H, 2009 28 days Mice MaleMoragrega , 2003 30 days Mice MaleLiu X, 2013 42 days Mice MaleWorkman JL, 2011 42 days Mice MaleVoikar V, 2005 49 days Mice MaleImanaka A, 2006 7 days Rats FemaleLeussis MP, 2008 7 days Rats FemaleDa Silva NL, 1997 28 days Rats FemalePisu MG, 2013 28 days Rats FemaleImanaka A, 2009 35 days Rats FemaleDoremus Fitzwater TL, 2009 49 days Rats FemaleKnuth ED, 2007 7 days Rats Female and MaleReis FM, 2012 0.02 days Rats MaleReis FM, 2012 0.08 days Rats MaleReis FM, 2012 1 days Rats MaleHaller J, 1999 4 days Rats MaleCheeta S, 2001 7 days Rats MaleCheeta S, 2001 7 days Rats MaleCheeta S, 2001 7 days Rats MaleHaller J, 2000 7 days Rats MaleHaller J, 2000 7 days Rats MaleLeussis MP, 2008 7 days Rats MaleMajercsik, 2003 7 days Rats MaleMajercsik, 2003 7 days Rats MaleMajercsik, 2003 7 days Rats MaleReis FM, 2012 7 days Rats MaleWright, 2001 7 days Rats MaleDas SK, 2015 14 days Rats Maledos Santos L, 2010 14 days Rats MaleLapiz MD, 2001 14 days Rats MaleLapiz MD, 2001 14 days Rats MaleQuintino dos Santos JW, 2014 20 days Rats MaleBledsoe AC, 2011 21 days Rats MaleBrenes JC, 2009 28 days Rats MaleDa Silva NL, 1996 28 days Rats MalePisu MG, 2011 28 days Rats MalePisu MG, 2014 28 days Rats MaleSerra M, 2000 28 days Rats MaleCuenya L, 2012 30 days Rats MaleFone KC, 1996 35 days Rats MaleFone KC, 1996 35 days Rats MaleImanaka A, 2008 35 days Rats MaleChappell AM, 2013 42 days Rats MaleHirani K, 2005 42 days Rats MaleKokare DM, 2010 42 days Rats MaleMcCool BA, 2009 42 days Rats MalePritchard LM, 2008 42 days Rats MaleRyu V, 2009 42 days Rats MaleDoremus Fitzwater TL, 2009 49 days Rats MaleYildirim E, 2012 49 days Rats MaleYorgason JT, 2013 49 days Rats MaleZhang Y, 2012 56 days Rats MaleThorsell A, 2006 84 days Rats Male

Wei XY, 2007 21 days Mice MaleVoikar V, 2005 49 days Mice MaleVoikar V, 2005 49 days Mice MaleCarrier N, 2012 21 days Rats FemaleCarrier N, 2012 21 days Rats MaleMcCool BA, 2009 42 days Rats Male

Linge R, 2013 28 days Mice MaleLiu X, 2013 42 days Mice MaleConrad KL, 2011 49 days Mice MaleKnuth ED, 2007 7 days Rats FemaleHermes G, 2011 70 days Rats FemaleKnuth ED, 2007 7 days Rats MaleBlakley G, 2006 14 days Rats MaleDas SK, 2015 14 days Rats MaleDjordjevic J, 2012 21 days Rats MaleLukkes JL, 2009 21 days Rats MaleThorsell A, 2006 84 days Rats Male

N

1114

889

85

140

10 32 10 10 10 10 14 97 14 10 14 10 10 10 32 10 10 15 15 8 10 27 12 8 18 14 14 14 9 7 7 7 9 12 8 9 9 9 14 5 12 12 6 6 18 12 12 10 18 20 13 10 5 5 12 16 8 7 28 6 36 8 10 7 10 9

9 15 15 9 9 28

10 10 10 8 8 9 33 12 18 12 10

Mean

5.4010.5814.9522.1219.4315.47 4.6715.08 4.67 2.71 4.6729.2513.4141.8038.10 8.0931.46 2.4324.3716.1743.8919.2425.80 3.94 0.0627.9427.9427.9413.8336.5733.6221.96 7.0614.46 7.6314.3612.66 9.7527.9410.5728.51 9.0829.7517.1916.1013.31 7.6838.9219.8912.7730.92 9.9710.5812.4718.5723.6218.4713.5521.5023.8812.83 6.3027.3420.7713.1018.40

47.6243.1046.4031.6129.4540.47

13.6522.1323.43 7.2243.63 6.77 0.5020.4816.9748.0911.31

Group housed

SD

18.41 9.3110.97 6.58 7.24 6.96 2.9918.53 2.9919.34 2.9915.78 7.8127.6116.9753.4920.0917.0432.7945.7323.4131.1017.6723.7076.8323.7723.7723.7721.1717.11 9.4713.50 3.1713.8545.9012.1213.20 8.1023.7712.22 6.32 7.3314.40 7.7929.9048.84 1.4362.3968.7639.4441.87 4.4628.3633.0814.2041.14 6.4320.5356.0912.0880.3068.8520.4311.19 2.7820.11

17.9922.0817.8227.4721.1647.62

25.6535.6721.1319.5220.2915.00 3.10 4.4829.9223.2213.39

N

1109

906

89

114

10 40 10 10 10 10 14 94 14 10 14 10 11 12 33 10 10 15 12 8 10 26 13 8 18 13 12 13 9 8 8 8 9 12 8 9 9 9 14 5 8 12 6 6 27 12 12 10 18 20 13 11 5 5 16 17 8 7 31 6 36 8 10 7 10 7

10 15 15 9 9 31

10 10 10 10 8 9 10 8 18 12 9

Mean

3.03 6.9417.5015.4022.1313.42 4.83 4.36 5.53 8.00 6.0011.07 8.2014.1139.40 7.1217.5015.9722.50 4.8820.03 7.5514.27 5.36 0.0422.1812.33 6.57 2.7029.6931.6631.2411.8810.35 7.29 8.62 4.81 6.8211.23 5.5323.24 2.1217.17 9.8218.39 6.43 4.5118.18 1.1812.6813.96 5.3814.9324.1416.13 8.27 9.28 6.2815.6733.85 4.96 1.6029.81 7.6512.1039.20

26.2242.2031.0017.3514.0142.40

4.19 5.9312.04 7.0819.52 4.57 0.6716.8411.3834.9220.04

Isolated

SD

18.9610.4110.3713.6011.6114.70 2.6263.27 4.2437.53 3.12 4.84 5.5612.2214.3649.05 8.9259.6420.3728.2739.0019.7315.8624.0746.9015.2614.63 7.63 5.2911.89 9.8523.2310.23 8.5537.92 8.61 6.60 4.8915.84 4.99 4.29 2.45 6.34 2.7156.6345.16 1.1246.79 8.9640.96 1.27 2.6326.8722.8121.6028.26 6.1514.9051.0913.8242.2512.9733.19 9.33 5.1921.16

18.2214.3319.7515.8417.9731.18

11.1517.8422.2424.6716.6314.40 5.31 2.9737.4213.0423.64

2 0 2 4


Standardised mean differenceSMD

3e 01

3e 01

4e 01

3e 01

1e 01 4e 012e 01

6e 013e 01

2e 016e 02

2e 012e 012e 014e 01

1e+00 7e 01 1e+008e 02

2e 02 9e 013e 01

6e 02 3e 01 7e 01 4e 01 7e 016e 02

3e 04 3e 01 8e 01 1e+00 7e 01 4e 01 2e 015e 016e 01

3e 01 8e 03 5e 01 7e 01 4e 01 8e 01 5e 01 9e 01 1e+00 1e+00 1e+005e 02

1e 01 2e+00 4e 01 4e 01 2e 03 6e 01 1e+001e 014e 01

1e 01 4e 01 1e+00 4e 01 1e 017e 01

1e 01 9e 029e 02

1e+00 2e 011e+00

1e+00 5e 02 8e 01 6e 01 7e 015e 02

5e 01 6e 01 5e 01 6e 03 1e+00 1e 014e 02

9e 01 2e 01 7e 014e 01

95% CI

[ 0.21; 0.44]

[ 0.18; 0.45]

[ 0.06; 0.75]

[ 0.06; 0.59]

[ 0.76; 1.00][ 0.11; 0.83][ 1.11; 0.65][ 0.30; 1.50][ 1.15; 0.61][ 0.71; 1.05][ 0.80; 0.68][ 0.05; 0.52][ 0.97; 0.51][ 1.05; 0.71][ 1.17; 0.33][ 0.48; 2.51][ 0.15; 1.64][ 0.35; 2.23][ 0.57; 0.40][ 0.86; 0.89][ 0.07; 1.78][ 1.02; 0.42][ 0.69; 0.82][ 0.71; 1.27][ 0.20; 1.62][ 0.11; 0.99][ 0.14; 1.48][ 1.04; 0.92][ 0.65; 0.65][ 0.48; 1.04][ 0.05; 1.55][ 0.33; 1.98][ 0.27; 1.64][ 0.59; 1.48][ 0.83; 1.21][ 1.48; 0.58][ 1.56; 0.34][ 0.46; 1.15][ 0.97; 0.99][ 0.42; 1.46][ 0.24; 1.68][ 0.52; 1.35][ 0.03; 1.58][ 0.78; 1.76][ 0.05; 1.85][ 0.35; 2.12][ 0.19; 2.28][ 0.10; 2.43][ 0.64; 0.55][ 0.66; 0.94][ 1.30; 3.47][ 0.53; 1.25][ 0.29; 1.03][ 0.62; 0.62][ 0.23; 1.34][ 0.27; 2.16][ 1.38; 1.10][ 1.63; 0.89][ 0.62; 0.87][ 0.26; 1.12][ 0.26; 2.50][ 0.68; 1.44][ 0.40; 0.62][ 1.89; 0.47][ 0.34; 0.58][ 0.89; 1.07][ 0.96; 0.79][ 0.02; 2.36][ 0.65; 1.11][ 2.01; 0.10]

[ 0.14; 2.11][ 0.67; 0.76][ 0.05; 1.54][ 0.34; 1.56][ 0.21; 1.71][ 0.56; 0.46]

[ 0.43; 1.35][ 0.35; 1.45][ 0.39; 1.40][ 0.92; 0.94][ 0.13; 2.32][ 0.78; 1.07][ 0.75; 0.66][ 0.06; 1.83][ 0.49; 0.82][ 0.15; 1.50][ 1.35; 0.47]

W(random)

100%

79.5%

7.8%

12.7%

1.1% 2.1% 1.1% 1.1% 1.1% 1.1% 1.4% 2.7% 1.4% 1.1% 1.4% 0.9% 1.1% 1.0% 2.1% 1.1% 1.1% 1.4% 1.4% 1.0% 1.1% 1.9% 1.3% 1.0% 1.6% 1.4% 1.3% 1.2% 1.0% 0.9% 0.9% 0.9% 1.0% 1.3% 1.0% 1.0% 1.0% 1.0% 1.3% 0.7% 1.0% 1.1% 0.7% 0.7% 1.7% 1.3% 0.8% 1.1% 1.6% 1.7% 1.3% 1.0% 0.7% 0.7% 1.4% 1.5% 0.8% 0.9% 2.0% 0.7% 2.1% 1.0% 1.1% 0.8% 1.1% 0.9%

1.0% 1.5% 1.4% 1.0% 1.0% 2.0%

1.1% 1.1% 1.1% 1.1% 0.8% 1.1% 1.5% 1.0% 1.6% 1.2% 1.1%

Figure 7. Meta-analyses of experiments on the stress-anxiety relationship in rodents.

Meta-analysis of effect sizes of stress-anxiety interventions is shown as a forest plot of standardized effect sizes (Hedges’ g). Error bars indicate the 95% confidence intervals of g. The weighted average meaneffect size of all studies is represented by the central vertices of a red diamond; the outer vertices indicate the 95% confidence intervals. Control and treatment samples sizes (N), and the assay types of the studies are given; elevated plus maze (EPM), open field (OF) and light-dark box (LD). Effects of: A. Acute pain. B. Physical restraint (immobilisation). C. Social isolation.

A

B

C

Pain



Assay = EPM

Assay = LD

Assay = OF







Shang, 2015 1 days Mice Male EPMShang, 2015 3 days Mice Male EPMShang, 2015 7 days Mice Male EPMShang, 2015 14 days Mice Male EPMLiu, 2015 12 days Mice Male EPMLiu, 2015 12 days Mice Male EPMParent, 2012 28 days Rats Male EPMLeite Almeida, 2009 30 days Rats Male EPMBenbouzid, 2008 30 days Mice Male EPMMatsuzawa Yanagida, 2008 28 days Mice Male EPMLeite Almeida, 2009 30 days Rats Male EPMSchellinck, 2003 0 days Mice Female and Male EPM

Parent, 2012 28 days Rats Male LDMatsuzawa Yanagida, 2008 28 days Mice Male LD

Shang, 2015 1 days Mice Male OFShang, 2015 3 days Mice Male OFShang, 2015 7 days Mice Male OFShang, 2015 14 days Mice Male OFLiu, 2015 12 days Mice Male OFLiu, 2015 12 days Mice Male OFParent, 2012 28 days Rats Male OF

N

126

23

56

9 9 13 14 8 10 9 12 6 18 12 6

5 18

7 6 10 13 7 8 5

Mean

8.72 9.7315.6217.9884.6475.4340.8126.4333.9435.9126.4348.80

23.1024.55

4.51 4.45 4.11 3.6030.6812.45 4.99

SD

6.30 5.89 9.3811.0631.5234.5621.2121.9111.33 5.4721.91 8.64

10.69 6.30

3.92 2.38 1.71 2.6911.46 4.08 2.71

205

Control

2 0 2 4



0.56

0.72

0.58

0.22

0.63 0.07 0.34 0.330.21

0.13 1.05 1.24 1.89 2.22 0.03 1.66

0.91 1.98

0.06 0.13 0.61 0.58 0.721.02

0.33

95% CI

[ 0.19; 0.93]

[ 0.27; 1.17]

[ 2.24; 3.41]

[ 0.21; 0.66]

[ 0.30; 1.56][ 0.92; 1.05][ 0.41; 1.08][ 0.40; 1.07][ 1.14; 0.72][ 0.65; 0.92][ 0.05; 2.05][ 0.36; 2.13][ 0.55; 3.23][ 1.37; 3.07][ 0.77; 0.83][ 0.27; 3.05]

[ 2.25; 0.43][ 1.16; 2.79]

[ 1.08; 0.95][ 0.93; 1.19][ 0.29; 1.51][ 0.22; 1.38][ 0.28; 1.72][ 2.08; 0.04][ 0.92; 1.58]

W(random)

100%

58.5%

9.0%

32.5%

4.9% 4.7% 5.5% 5.6% 4.9% 5.4% 4.7% 5.0% 3.7% 5.2% 5.3% 3.6%

3.7% 5.3%

4.6% 4.5% 5.0% 5.3% 4.7% 4.5% 3.9%

223

Treatment

N

139

23

61

10 7 15 15 10 17 9 12 8 18 12 6

5 18

8 8 10 12 10 8 5

Mean

3.84 8.90 8.9711.0392.5771.3620.71 5.9717.5622.4125.7928.93

31.6514.90

4.96 3.65 1.98 1.6923.4621.53 2.04

SD

8.2316.6724.6726.0039.3026.5114.85 4.92 4.60 6.4025.1013.01

5.42 2.43

8.82 7.38 4.40 3.64 7.9911.2311.14


https://doi.org/10.1101/020701


Figure 8: Interventions with no effect on anxiety-related behaviors. Meta-analysis of effect sizes of non-anxiety-related interventions, shown as a forest plot of standardized effect sizes (Hedges’ g). Error bars indicate the 95% confidence intervals of g. The weighted average mean effect size of all studies is represented by the central vertices of a red diamond; the outer vertices indicate the 95% confidence intervals. Control and treatment samples sizes (NC, NT) and the assay types of the studies are given; elevated plus maze (EPM), open field (OF) and light-dark box (LD). Effects of: A. Adcy1 gene knockout models. B. Synaptotagmin gene (Syt1) mutation models.

AC1 KO g = 0.24, p = 0.28, I2 = 0 %

Reference Nc Nt Assay

Krishnan V 2008

Storm DR 1998

Krishnan V 2008

Krishnan V 2008

10

8

10

10

11

8

11

11

OF

OF

LD

EPM

A−2 −1 0 1 2

Syt1 KO g = −0.25, p = 0.27, I2 = 0 %

−2.0 −1.5 −1.0 −0.5 0.0 0.5 1.0

Difference from controls (SD)

Reference Nc Nt Assay

Powell CM 2004

Powell CM 2004

24

1520

19

LD

EPM

B

Difference from controls (SD)


https://doi.org/10.1101/020701


Figure 9. Summary effect sizes of all meta-analyses. The weighted mean effect sizes

of all 12 interventions are shown here. Each mean effect size is represented by the central

vertices of a diamond; the outer vertices indicate the 95% confidence intervals. The hori-

g, the standard deviation change relative to control animals. Color

indicates direction (green = anxiolytic, red = anxiogenic) and statistical significance (grey

= statistically non-significant). The diamonds for the diazepam, social isolation, and Htt KO

meta-analyses represent the summary effect sizes after trim-and-fill bias correction.

Pain

-1.5 -1 -0.5 0 0.5 1 1.5

Crhr1 KO

Htt OE

Diazepam

Ht1a OE

Syt KO

Ac1 KO

Crh KO

Htt KO

Restraint

Htr1a KO

Isolation

Hedges’ gAnxiolytic Anxiogenic


https://doi.org/10.1101/020701


Table 1. Summary of systematic reviews of anxiety-related interventions in mouse and rat. The PubMed query phrases used to identify articles that might contain data relevant to the interventions and assays of interest are detailed. Title, abstract and full-text searches were performed to identify articles meeting the selected criteria. Where there were fewer than ten articles meeting the selection criteria, all relevant data in all articles found were used for data extraction and meta-analysis. Where there were more articles, we took a random sample of ten papers and used only relevant data therein for meta-analysis.Intervention

Table 1. Summary of systematic reviews of anxiety-related interventions in mouse and rat. The PubMed query phrases used to identify articles that might contain data relevant to the interventions and assays of interest are detailed. Title, abstract and full-text searches were performed to identify articles meeting the selected criteria. Where there were fewer than ten articles meeting the selection criteria, all relevant data in all articles found were used for data extraction and meta-analysis. Where there were more articles, we took a random sample of ten papers and used only relevant data therein for meta-analysis.




Query phrase used in PubMed for study selection Articles identified

by phrase

Articles meeting criteria

Articles in meta-analysis

Diazepam

Ht1a knockout

Ht1a over-expression

Htt knockout

Htt over-expression

Pain

Restraint

Isolation

Adcy1 knockout

Synaptotagmin

540 172 172

(serotonin1A receptor OR 5-HT1A receptor AND knockout AND anxiety)

85 12 12

(serotonin1A receptor) OR 5-HT1A receptor) AND (over-expression OR over-expression OR over-expressing) AND anxiety

13 3 3

(serotonin transporter knockout OR knockdown OR deletion OR antisense AND anxiety AND (elevated plus maze OR open field OR light-dark) AND (rats OR rat OR mice OR mouse OR Mus)

37 13 13

(serotonin transporter AND anxiety AND (elevated plus maze OR open field OR light-dark) AND (increased OR over-expression OR overexpressing OR transgenic)) AND (rats OR rat OR mice OR mouse OR Mus)

65 2 2

(Inflammatory pain AND anxiety OR neuropathic pain AND anxiety AND (elevated plus maze OR open field OR light-dark))) AND (rats OR rat OR mice OR mouse OR Mus)

49 7 7

(acute restraint AND anxiety AND (elevated plus maze OR open field OR light-dark) AND (rats OR rat OR mice OR mouse OR Mus)

87 15 15

(social Isolation OR single housing)) AND anxiety) AND (elevated plus maze OR plus-maze OR open field OR OFT OR light-dark)) AND (rats OR rat OR mice OR mouse OR Mus)

155 50 50

Ad hoc literature search 2 2 2

Ad hoc literature search 1 1 1

Crh knockout

Crhr1 knockout

(CRF OR CRH OR Corticotropin releasing factor AND knockout AND anxiety AND (elevated plus maze OR open field OR light-dark))) AND (rats OR rat OR mice OR mouse OR Mus)

62 2 2

(Corticotropin releasing factor receptor 1-deficient mice) OR CRH1 receptor antisense oligodeoxynucleotide OR Crhr1 null mutants OR Corticotropin-releasing hormone receptor antisense)

62 6 6

((((diazepam OR valium)) AND anxiety) AND (open field OR exploratory)) AND (rodent OR rat OR rats ORmouse OR mice OR Mus)


https://doi.org/10.1101/020701


Table 2. Summary of Meta-Analyses of Ten Anxiety-related Interventions and Two

Non-anxiety-related Interventions in Rodents

Shown here are the summary effect sizes (Hedges’ g) for the ten ARDEBs reviewed, along-side 2 non-ARDEB-related interventions (Ac1 KO and Syt KO) in rodents. I2 — Heterogeneity of mixed-effects meta-analysis. KO — knockout, OE — Over-expression. Meta-analyses with more than 10 experiments were tested for publication bias with Egger’s test. 95% confidence intervals (95% CI) are given in square brackers for Hedges’ g (adjusted and unadjusted), as well as for I2.

Experiments

386Diazepam

No. of

Studies

83Isolation

Hedges' g[95% CI]

29Htt KO

p-value

21Restraint

I2

[95% CI]

21Pain

Egger's Test

p-value

20Ht1a KO

No. of

Experiments Added

After Trim-and-Fill

8Ht1a OE

Adjusted Hedges' g[95% CI]

8Crhr1 KO

p-value

Adjusted Hedges' g

4Htt OE 4Ac1 KO 2Crh KO 2Syt KO

17250

13167

11362221

-1.3 [-1.4; -1.2]0.33 [0.21; 0.44]0.88 [0.65; 1.1]

0.67 [0.0062; 1.3]0.56 [0.19; 0.92]

0.73 [0.5; 0.96]

-0.6 [-1.3; 0.13]-1 [-1.3; -0.7]

-0.94 [-1.7; -0.2]

0.24 [-0.2; 0.69]

0.3 [-0.32; 0.92]

-0.25 [-0.7; 0.2]

1.36e-1443.37e-085.18e-14

0.0479

0.002833.15e-10

0.105

6.64e-110.01270.2840.34

0.268

70.4 [67.2; 73.3]40.7 [3.5; 44.6]

54.6 [23.7; 67.6]

89.3 [75.8; 88.7]

68.1 [44.6; 78]46.6 [0; 58.2]

82.7 [61.8; 89.8]

0 [0; 55.7]

46.8 [0; 83.8]0 [0; 75.9]

0 [NA; NA]0 [NA; NA]

2.35e-56

0.01466.72e-060.0305

0.7630.545

NANANANANANA

10212100

NANANANANANANANA

-0.85 [-0.96; -0.74]

0.21 [0.069; 0.34]

0.57 [0.29; 0.86]

NANANANANANANANA

4.72e-56

0.003169.71e-05

NANANANANANANANA

0.67 [0.0062; 1.3] 0.0479

No. of


https://doi.org/10.1101/020701


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