Protocol for a systematic review and meta-analysis of data from preclinical studies employing forced swimming test: an update.

OBJECTIVE: Forced swimming test (FST) in rodents is a widely used behavioural test for screening antidepressants in preclinical research. Translational value of preclinical studies may be improved by appraisal of the quality of experimental design and risk of biases, which remains to be addressed for FST. The present protocol of a systematic review with meta-analysis aims to investigate the quality of preclinical studies employing FST to identify risks of bias in future publications. In addition, this protocol will help to determine the effect sizes (ES) for primary and secondary outcomes according to several aspects of the FST study design. SEARCH STRATEGY SCREENING ANNOTATION DATA MANAGEMENT: Publications reporting studies testing different classes of antidepressants in FST will be collected from Medline, SCOPUS and Web of Science databases. A broad list of inclusion criteria will be applied excluding those studies whereby FST is used as a stressor or studies reporting data from co-treatments. For assessing the quality of the included publications, the quality checklist adapted by Collaborative Approach to Meta-Analysis and Review of Animal Data from Experimental Studies will be used. If the meta-analysis seems feasible, the ES and the 95% CI will be analysed. The heterogeneity between studies will be assessed by using the χ2statistic with n-1 degrees of freedom. Subgroup meta-analysis (meta-regression, and if necessary, stratified regression) will be performed when possible according to characteristics of study design and study quality to assess their impact on efficacy of the treatments. In addition, funnel plotting, Egger regression, and 'trim and fill' will be used to assess the risk of publication bias. Results of this protocol will help to create rational methodological guidelines for application of FST in rodents and improve the quality and translational value of preclinical research on antidepressant discovery. REPORTING: A preliminary version of the present protocol has been preregistered with Systematic Review Facility (http://syrf.org.uk/). A preprint version of the current protocol has been registered with Open Science Framework (https://osf.io/9kxm4/). Results will be communicated in scientific meetings and peer-reviewed journals. We plan to conduct an anonymous and online survey within the scientific community to ask researchers about their perception of risk of bias and their experience with the publication of negative results.

The mesencephalic GCt-ICo complex and tonic immobility in pigeons (Columba livia): a c-Fos study.

Tonic immobility (TI) is a response to a predator attack, or other inescapable danger, characterized by immobility, analgesia and unresponsiveness to external stimuli. In mammals, the periaqueductal gray (PAG) and deep tectal regions control the expression of TI as well as other defensive behaviors. In birds, little is known about the mesencephalic circuitry involved in the control of TI. Here, adult pigeons (both sex, n = 4/group), randomly assigned to non-handled, handled or TI groups, were killed 90 min after manipulations and the brains processed for detection of c-Fos immunoreactive cells (c-Fos-ir, marker for neural activity) in the mesencephalic central gray (GCt) and the adjacent nucleus intercollicularis (ICo). The NADPH-diaphorase staining delineated the boundaries of the sub nuclei in the ICo-GCt complex. Compared to non-handled, TI (but not handling) induced c-Fos-ir in NADPH-diaphorase-rich and -poor regions. After TI, the number of c-Fos-ir increased in the caudal and intermediate areas of the ICo (but not in the GCt), throughout the rostrocaudal axis of the dorsal stratum griseum periventriculare (SGPd) of the optic tectum and in the n. mesencephalicus lateralis pars dorsalis (MLd), which is part of the ascending auditory pathway. These data suggest that inescapable threatening stimuli such as TI may recruit neurons in discrete areas of ICo-GCt complex, deep tectal layer and in ascending auditory circuits that may control the expression of defensive behaviors in pigeons. Additionally, data indicate that the contiguous deep tectal SCPd (but not GCt) in birds may be functionally comparable to the mammalian dorsal PAG.

Defensive behaviors and prosencephalic neurogenesis in pigeons (Columba livia) are affected by environmental enrichment in adulthood.

Neurogenesis in the adult brain appears to be phylogenetically conserved across the animal kingdom. In pigeons and other adult non-oscine birds, immature neurons are observed in several prosencephalic areas, suggesting that neurogenesis may participate in the control of different behaviors. The mechanisms controlling neurogenesis and its relevance to defensive behaviors in non-oscine birds remain elusive. Herein, the contribution of the environment to behavior and neurogenesis of pigeons was investigated. Adult pigeons (Columba livia, n = 6/group), housed in standard (SE) or enriched environment (EE) for 42 days, were exposed to an unfamiliar environment (UE) followed by presentation to a novel object (NO). Video recordings of UE+NO tests were analyzed and scored for latency, duration and frequency of angular head movements, peeping, grooming, immobility and locomotion. Twenty-four hours later, pigeons were submitted to the tonic immobility test (TI) and number of trials for TI and TI duration were scored, followed by euthanasia 2 h later. Brains were immunohistochemically processed to reveal doublecortin (DCX), a marker for newborn neurons. Compared to those housed in SE, the pigeons housed in EE responded to a NO with more immobility. In addition, the pigeons housed in EE presented longer TI, more DCX-immunoreactive (DCX-ir) cells in the hippocampus and fewer DCX-ir cells in the lateral striatum than those housed in SE. There was no correlation between the number of DCX-ir cells and the scores of immobility in behavioral tests. Together, these data suggest that enrichment favored behavioral inhibition and neurogenesis in the adult pigeons through different, parallel mechanisms.

Distribution and characterization of doublecortin-expressing cells and fibers in the brain of the adult pigeon (Columba livia).

Doublecortin (DCX) is a microtubule-associated protein essential for the migration of immature neurons in the developing and adult vertebrate brain. Herein, the distribution of DCX-immunoreactive (DCX-ir) cells in the prosencephalon of the adult pigeon (Columba livia) is described, in order to collect the evidence of their immature neural phenotype and to investigate their putative place of origin. Bipolar and multipolar DCX-ir cells were observed to be widespread throughout the parenchyma of the adult pigeon forebrain. Small, bipolar and fusiform DCX-ir cells were especially concentrated at the tips of the lateral walls of the lateral ventricles (VZ) and sparsely distributed in the remaining ependyma. Multipolar DCX-ir cells populated the pallial regions. None of these DCX-ir cells seemed to co-express NeuN or GFAP, suggesting that they were immature neurons. Two different migratory-like routes of DCX-ir cells from the VZ toward different targets in the parenchyma were putatively identified: (i) rostral migratory-like bundle; and (ii) lateral migratory-like bundle. In addition, pial surface bundles and intra-ependymal fascicles were also observed. Pigeons treated with 5-bromo-desoxyuridine (BrdU, 3 intraperitoneal injections of 100mg/kg 2h apart, sacrificed 2h after last injection) displayed BrdU-immunoreactive cells (BrdU-ir) in VZ and ependyma whereas the parenchyma was free of such cells. Despite the regional overlapping, there was no evidence of double-labeling between BrdU and DCX. Therefore, the VZ in the brain of adult pigeons seems to have rapidly dividing cells as putative progenitors of newborn neurons populating the forebrain. The distribution of the newborn neurons in the avian prosencephalon and their migration pathways appear to be larger than in mammals, suggesting that the morphological turnover of forebrain circuits is an important mechanism for brain plasticity in avian species during adulthood.

Repeated rat-forced swim test: reducing the number of animals to evaluate gradual effects of antidepressants.

The forced swim test (FST) is a pre-clinical test to short and long term treatment with antidepressant drugs (ADT), which requires between-subject designs. Herein a modified protocol of the FST using within-subject design (repeated rat-FST) was evaluated. Male Wistar rats were submitted to 15 min of swimming (Day 1: pretest) followed by three subsequent 5 min-swimming tests one week apart (Day 2: test, Day 7: retest 1, Day 14: retest 2). To determine the temporal and factorial characteristics of the variables scored in the repeated rat-FST, the protocol was carried out in untreated animals (E1). To validate the method, daily injections of Fluoxetine (FLX, 2.5mg/kg, i.p.) or saline were given over a 2-week period (E2). Tests and retests have been videotaped for further register of the latency, frequency and duration of behaviors. Over retesting the latency to immobility decreased whereas duration of immobility tended to increase. Factorial analysis revealed that the test, the retest 1 as well as the retest 2 have variables suitable to detection of antidepressant-like effects of ADT. Compared to saline, FLX chronically administrated reduced duration of immobility whereas increased duration of swimming in retest 2. The data suggest that repeated rat-FST detected the gradual increase in the efficacy of low doses of FLX over time. Therefore, repeated rat-FST seemed suitable to detect short and long term effects of selective serotonin reuptake inhibitors, or other ADT, thus reducing the number of animals used in the screenings of this type of compounds.

Eag 1, Eag 2 and Kcnn3 gene brain expression of isolated reared rats.

The Eag1 and Eag2, voltage-dependent potassium channels, and the small-conductance calcium-activated potassium channel (Kcnn3) are highly expressed in limbic regions of the brain, where their function is still unknown. Eag1 co-localizes with tyrosine hydroxilase enzyme in the substantia nigra and ventral tegmental area. Kcnn3 deficiency leads to enhanced serotonergic and dopaminergic neurotransmission accompanied by distinct alterations in emotional behaviors. As exposure to stress is able to change the expression and function of several ion channels, suggesting that they might be involved in the consequences of stress, we aimed at investigating Eag 1, Eag2 and Kcnn3 mRNA expression in the brains of rats submitted to isolation rearing. As the long-lasting alterations in emotional and behavioral regulation after stress have been related to changes in serotonergic neurotransmission, expressions of serotonin Htr1a and Htr2a receptors in male Wistar rats' brain were also investigated. Rats were reared in isolation or in groups of five for nine weeks after weaning. Isolated and socially reared rats were tested for exploratory activity in the open field test for 5 min and brains were processed for reverse-transcription coupled to quantitative polymerase chain reaction (qRT-PCR). Isolated reared rats showed decreased exploratory activity in the open field. Compared to socially reared rats, isolated rats showed reduced Htr2a mRNA expression in the striatum and brainstem and reduced Eag2 mRNA expression in all examined regions except cerebellum. To our knowledge, this is the first work to show that isolation rearing can change Eag2 gene expression in the brain. The involvement of this channel in stress-related behaviors is discussed.

Eag1 potassium channel immunohistochemistry in the CNS of adult rat and selected regions of human brain.

Eag1 (K(V)10.1) is the founding member of an evolutionarily conserved superfamily of voltage-gated K(+) channels. In rats and humans Eag1 is preferentially expressed in adult brain but its regional distribution has only been studied at mRNA level and only in the rat at high resolution. The main aim of the present study is to describe the distribution of Eag1 protein in adult rat brain in comparison to selected regions of the human adult brain. The distribution of Eag1 protein was assessed using alkaline-phosphatase based immunohistochemistry. Eag1 immunoreactivity was widespread, although selective, throughout rat brain, especially noticeable in the perinuclear space of cells and proximal regions of the extensions, both in rat and human brain. To relate the results to the relative abundance of Eag1 transcripts in different regions of rat brain a reverse-transcription coupled to quantitative polymerase chain reaction (real time PCR) was performed. This real time PCR analysis showed high Eag1 expression in the olfactory bulb, cerebral cortex, hippocampus, hypothalamus, and cerebellum. The results indicate that Eag1 protein expression greatly overlaps with mRNA distribution in rats and humans. The physiological relevance of potassium channels in the different regions expressing Eag1 protein is discussed.

Effects of acute and chronic fluoxetine treatments on restraint stress-induced Fos expression.

Chronic treatment with antidepressants has been shown to attenuate behavioral changes induced by uncontrollable stress. The mechanisms and brain sites of this effect, however, remain controversial. The objective of the present work was to investigate the effects of chronic and acute treatment with fluoxetine (FLX), a selective serotonin reuptake blocker, on Fos expression in animals submitted to restraint stress. Male Wistar rats (n = 3-9/group) received, during 1 or 21 days, intraperitoneal. Injections of vehicle (saline + 0.2% Tween-80, 1 ml/kg) or FLX (10 mg/kg). One hour after the last injection they were forced restrained for 2 h and sacrificed immediately after. Non-stressed animals were sacrificed 2 h after the last injection. The brains were removed and processed for immunohistochemistry. Fos-like immunoreactivity (FLI) was quantified by a computer system. In acutely treated animals FLX decreased stress-induced FLI in the medial amygdala (MeA), bed nucleus of the stria terminalis (BNST), ventrolateral part, and dorsolateral periaqueductal gray (PAG). After chronic treatment, however, the drug induced a significant increase in FLI in the BNST (ventrolateral and medial parts), lateral septal nucleus (LSN, dorsal part), dorsal raphe nucleus (DRN), and locus coeruleus in restrained group. In non-restrained animals chronic treatment with FLX increased FLI in the MeA, BNST (ventrolateral and dorsolateral parts), LSN (dorsal and intermediate parts), dorsolateral and dorsomedial PAG and in the DRN. The results suggest that chronic fluoxetine treatment induce plastic changes that result in a different regional pattern of Fos expression.

c-jun mRNA expression in the hippocampal formation induced by restraint stress.

The effect of restraint stress on c-jun mRNA expression in the hippocampal formation was investigated by in situ hybridization, dot blot and northern blot. c-jun mRNA expression increased after 60 min of forced restraint in the dentate gyrus, CA1 and CA3 regions of the hippocampal formation. The effect in the dentate gyrus was attenuated by pre-stress i.c.v. injection of the anxiolytic benzodiazepine midazolam (20 nmol/2 microl) or the N-methyl-D-aspartate (NMDA) receptor antagonist 2-amino-7-phosphonoheptanoic acid (AP-7, 5 nmol/2 microl), but not by the 5-HT1A agonist, (+/-) 8-hydroxy-dipropylaminotetralin (8-OH-DPAT, 20 nmol/2 microl). These results suggest that the hippocampal formation is activated during restraint stress, and that this activation is modulated by benzodiazepine/GABA-A or NMDA receptors.