Exploring the similarities between risk factors triggering depression in humans and elevated in-cage "inactive but awake" behavior in laboratory mice.

Introduction: Depression is a human mental disorder that can also be inferred in non-human animals. This study explored whether time spent inactive but awake ("IBA") in the home-cage in mice was further triggered by risk factors similar to those increasing vulnerability to depression in humans (early life stress, genetic predispositions, adulthood stress). Methods: Eighteen DBA/2 J and 18 C57BL/6 J females were tested, of which half underwent as pups a daily maternal separation on post-natal days 2-14 (early-life stress "ELS") (other half left undisturbed). To assess the effect of the procedure, the time the dams from which the 18 subjects were born spent active in the nest (proxy for maternal behavior) was recorded on post-natal days 2, 6, 10 and 14 for 1 h before separation and following reunion (matched times for controls), using live instantaneous scan sampling (total: 96 scans/dam). For each ELS condition, about half of the pups were housed post-weaning (i.e., from 27 days old on average) in either barren (triggering IBA and depression-like symptoms) or larger, highly enriched cages (n = 4-5 per group). Time mice spent IBA post-weaning was observed blind to ELS treatment using live instantaneous scan sampling in two daily 90-min blocks, two days/week, for 6 weeks (total: 192 scans/mouse). Data were analyzed in R using generalized linear mixed models. Results: The dams were significantly more active in the nest over time (p = 0.016), however with no significant difference between strains (p = 0.18), ELS conditions (p = 0.20) and before/after separation (p = 0.83). As predicted, post-weaning barren cages triggered significantly more time spent IBA in mice than enriched cages (p < 0.0001). However, neither ELS (p = 0.4) nor strain (p = 0.84) significantly influenced time mice spent IBA, with no significant interaction with environmental condition (ELS × environment: p = 0.2861; strain × environment: p = 0.5713). Discussion: Our results therefore only partly support the hypothesis that greater time spent IBA in mice is triggered by risk factors for human depression. We discuss possible explanations for this and further research directions.

Do greater levels of in-cage waking inactivity in laboratory mice reflect a spontaneous depression-like symptom? A pharmacological investigation.

We previously identified in laboratory mice an inactive state [being awake with eyes open motionless within the home cage; inactive but awake, 'IBA'] sharing etiological factors and symptoms with human clinical depression. We further test the hypothesis that greater time spent displaying IBA indicates a depression-like state in mice by investigating whether the antidepressant Venlafaxine, environmental enrichment, and their combination, alleviate IBA. Seventy-two C57BL/6J and 72 DBA/2J female mice were pseudo-randomly housed post-weaning in mixed strain-pairs in non-enriched (NE; 48 pairs) or in environmentally enriched (EE; 24 pairs) cages. After 34 days, half of the mice housed in NE cages were either relocated to EE cages or left in NE cages. For each of these conditions, half of the mice drank either a placebo or the antidepressant Venlafaxine (10 mg/kg). The 48 mice housed in EE cages were all relocated to NE cages and allocated to either the placebo (n = 24) or Venlafaxine (n = 24). IBA data were collected prior to and after environmental adjustment by trained observers blind to the pharmacological and environmental adjustment treatments. Data were analyzed using GLM models. NE cages triggered more IBA than EE cages (Likelihood-Ratio-Test Chi23 = 53.501, p < 0.0001). Venlafaxine and environmental enrichment appeared equally effective at reducing IBA (LRT Chi23 = 18.262, p < 0.001), and combining these approaches did not magnify their effects. Enrichment removal triggered IBA increase (LRT Chi21 = 23.050, p < 0.001), but Venlafaxine did not overcome the increase in IBA resulting from enrichment loss (LTR Chi21 = 0.081, p = 0.775). Theoretical implications for putative depression-like states in mice, and further research directions, are discussed.

Measuring affect-related cognitive bias: Do mice in opposite affective states react differently to negative and positive stimuli?

Affect-driven cognitive biases can be used as an indicator of affective (emotional) state. Since humans in negative affective states demonstrate greater responses to negatively-valenced stimuli, we investigated putative affect-related bias in mice by monitoring their response to unexpected, task-irrelevant stimuli of different valence. Thirty-one C57BL/6J and 31 DBA/2J females were individually trained to return to their home-cage in a runway. Mice then underwent an affective manipulation acutely inducing a negative (NegAff) or a comparatively less negative (CompLessNeg) affective state before immediately being tested in the runway with either an 'attractive' (familiar food) or 'threatening' (flashing light) stimulus. Mice were subsequently trained and tested again (same affective manipulation) with the alternative stimulus. As predicted, mice were slower to approach the light and spent more time with the food. DBA/2J mice were slower than C57BL/6J overall. Contrary to predictions, NegAff mice tended to approach both stimuli more readily than CompLessNeg mice, especially the light, and even more so for DBA/2Js. Although the stimuli successfully differentiated the response of mice to unexpected, task-irrelevant stimuli, further refinement may be required to disentangle the effects of affect manipulation and arousal on the response to valenced stimuli. The results also highlight the significant importance of considering strain differences when developing cognitive tasks.

The Period protein homolog LIN-42 negatively regulates microRNA biogenesis in C. elegans.

MicroRNAs (miRNAs) are small RNAs that post-transcriptionally regulate gene expression in many multicellular organisms. They are encoded in the genome and transcribed into primary (pri-) miRNAs before two processing steps that ultimately produce the mature miRNA. In order to generate the appropriate amount of a particular miRNA in the correct location at the correct time, proper regulation of miRNA biogenesis is essential. Here we identify the Period protein homolog LIN-42 as a new regulator of miRNA biogenesis in Caenorhabditis elegans. We mapped a spontaneous suppressor of the normally lethal let-7(n2853) allele to the lin-42 gene. Mutations in this allele (ap201) or a second lin-42 allele (n1089) caused increased mature let-7 miRNA levels at most time points when mature let-7 miRNA is normally expressed. Levels of pri-let-7 and a let-7 transcriptional reporter were also increased in lin-42(n1089) worms. These results indicate that LIN-42 normally represses pri-let-7 transcription and thus the accumulation of let-7 miRNA. This inhibition is not specific to let-7, as pri- and mature levels of lin-4 and miR-35 were also increased in lin-42 mutants. Furthermore, small RNA-seq analysis showed widespread increases in the levels of mature miRNAs in lin-42 mutants. Thus, we propose that the period protein homolog LIN-42 is a global regulator of miRNA biogenesis.

Functional genomic analysis of the let-7 regulatory network in Caenorhabditis elegans.

The let-7 microRNA (miRNA) regulates cellular differentiation across many animal species. Loss of let-7 activity causes abnormal development in Caenorhabditis elegans and unchecked cellular proliferation in human cells, which contributes to tumorigenesis. These defects are due to improper expression of protein-coding genes normally under let-7 regulation. While some direct targets of let-7 have been identified, the genome-wide effect of let-7 insufficiency in a developing animal has not been fully investigated. Here we report the results of molecular and genetic assays aimed at determining the global network of genes regulated by let-7 in C. elegans. By screening for mis-regulated genes that also contribute to let-7 mutant phenotypes, we derived a list of physiologically relevant potential targets of let-7 regulation. Twenty new suppressors of the rupturing vulva or extra seam cell division phenotypes characteristic of let-7 mutants emerged. Three of these genes, opt-2, prmt-1, and T27D12.1, were found to associate with Argonaute in a let-7-dependent manner and are likely novel direct targets of this miRNA. Overall, a complex network of genes with various activities is subject to let-7 regulation to coordinate developmental timing across tissues during worm development.

MicroRNA biogenesis: regulating the regulators.

MicroRNAs (miRNAs) function as 21-24 nucleotide guide RNAs that use partial base-pairing to recognize target messenger RNAs and repress their expression. As a large fraction of protein-coding genes are under miRNA control, production of the appropriate level of specific miRNAs at the right time and in the right place is integral to most gene regulatory pathways. MiRNA biogenesis initiates with transcription, followed by multiple processing steps to produce the mature miRNA. Every step of miRNA production is subject to regulation and disruption of these control mechanisms has been linked to numerous human diseases, where the balance between the expression of miRNAs and their targets becomes distorted. Here we review the basic steps of miRNA biogenesis and describe the various factors that control miRNA transcription, processing, and stability in animal cells. The tremendous effort put into producing the appropriate type and level of specific miRNAs underscores the critical role of these small RNAs in gene regulation.

Multiple cis-elements and trans-acting factors regulate dynamic spatio-temporal transcription of let-7 in Caenorhabditis elegans.

The let-7 microRNA (miRNA) is highly conserved across animal phyla and generally regulates cellular differentiation and developmental timing pathways. In Caenorhabditis elegans, the mature let-7 miRNA starts to accumulate in the last stages of larval development where it directs cellular differentiation programs required for adult fates. Here, we show that expression of the let-7 gene in C. elegans is under complex transcriptional control. The onset of let-7 transcription begins as early as the first larval stage in some tissues, and as late as the third larval stage in others, and is abrogated at the gravid adult stage. Transcription from two different start sites in the let-7 promoter oscillates during each larval stage. We show that transcription is regulated by two distinct cis-elements in the promoter of let-7, the previously described temporal regulatory element (TRE), and a novel element downstream of the TRE that we have named the let-7 transcription element (LTE). These elements play distinct and redundant roles in regulating let-7 expression in specific tissues. In the absence of the TRE and LTE, transcription of let-7 is undetectable and worms exhibit the lethal phenotype characteristic of let-7 null mutants. We also identify several genes that affect the transcription of let-7 generally and tissue-specifically. Overall, spatio-temporal regulation of let-7 transcription is orchestrated by multiple cis- and trans-acting factors to ensure appropriate expression of this essential miRNA during worm development.