Published On: Tue, Jan 8th, 2019

Improved dynamic monitoring of transcriptional activity during longitudinal analysis in the mouse brain [METHODS AND TECHNIQUES]

DISCUSSION

Using a combination of a modified lentiviral luciferase reporter and stereotaxic microinjection, we successfully demonstrated robust and dynamic brain GR activation in response to acute stress in mice. The main strength of this study is the achievement of dynamic monitoring of brain GR activity with a luciferase reporter tagged with destabilization motifs. Our strategy shows several other advantages over conventional methods. Importantly, it allows for anatomical pin-point analysis by stereotaxic microinjection of the reporter, which is applicable to animals other than mice. Moreover, the lentivirus-based transgene production of animal models is a much cheaper and more rapid process compared to the establishment of transgenic mice. The lentiviral-based transgene also shows long-term expression compared to an adenoviral-based transgene (Nayak and Herzog, 2010), and induces less of an inflammatory response than an adenovirus (Felizardo et al., 2011). Nevertheless, since the recombinant lentiviral vectors do still induce minimal inflammatory responses by removing over 95% of the parental viral genome, it is necessary to exert experimental caution by performing analysis of inflammation reactions.

We previously developed a lentivirus-based luciferase biosensor for the longitudinal analysis of GR activity. We monitored dynamic GR activation in the rat hippocampal CA1 after each 2-h exposure to acute stress after stereotaxic microinjection of the reporter. Although the reporter showed clear GR activation in the rat hippocampal CA1, the present results in murine IL-PFC showed only a weak signal of GR activation. Two possibilities for these observations can be proposed. The first is related to the different depth of bioluminescence signals since the dorsal hippocampal CA1 (anterior −1.88 mm, lateral −1.60 mm, ventral −1.30 mm) is deeper than the murine IL-PFC (anterior +1.95 mm, lateral −0.40 mm, ventral −3.10 mm). Indeed, bioluminescence is dependent on the physical constraints of light scattering and absorption in deep brain tissues (Aswendt et al., 2017). The second possibility is the differential expression pattern of GR. It is well-known that GR shows distinct expression patterns in different brain regions, although it is constitutively expressed (Vandevyver et al., 2014). As shown in Fig. 5, the GR expression pattern of hippocampal CA1 is denser than that of IL-PFC, which may influence the signal intensity derived from GR-expressing targeted cells.

Similar to previous findings (Leclerc et al., 2000; Li et al., 1998), a short half-life of luciferase was achieved by inserting the destabilizing sequence CP to the C-terminal of luciferase (in vitro T1/2=52.18±4.99 min). This short half-life resulted in high temporal resolution that was sufficient to discern a pattern of GR activity at 2-h time intervals of BLI during stress adaptation. In general, normal firefly luciferase has a half-life of 3–4 h, which can be suitable for studies requiring only once-daily assays over the course of several days, such as assessment of viral infection and treatment effects (Luker and Luker, 2009). This short half-life yielded poor signal intensities of luciferase, which has made it very difficult to study particularly deep brain regions. The further modified reporter described herein eliminates this disadvantage by replacing the Luc gene with Luc2, which is codon-optimized for expression in mammalian cells (Mašek et al., 2013). As expected, Luc2 yielded brighter bioluminescence signals induced by acute stress at 4 h post-stress. Unexpectedly, however, the destabilizing CP sequence significantly increased the bioluminescence signals as demonstrated by a 1.3-fold higher signal intensity of the Luc2CP reporter than that of the Luc2 reporter at 4 h post-stress. This suggests that the enhanced temporal resolution conferred by the destabilizing sequence CP allowed for episodic synchronization of GR activity, revealing an evident peak at 4 h post-stress. These results highlight the importance of achieving high temporal resolution for the dynamic monitoring of neurobiological processes in the brain.

Interestingly, our GRE-Luc reporter yielded weaker bioluminescence signals (∼1×105 p/s/cm2/sr in 1-min exposures), with signal intensity more than two orders of magnitude lower, in the brains compared with that reported for a Cre-transgenic mouse imaged using similar instrument settings (∼2×107 p/s/cm2/sr in 5-sec exposures; Akhmedov et al., 2016). This difference in signal intensity may be explained by different experimental conditions such as the different exposure time of BLI and the use of different transcription factor-binding arrays. However, the weak signal intensity may be related to the different animal model used in our study. We stereotactically injected a small amount of the reporter (1 μl) into the targeted brain region. Despite the relatively weaker signals, the intensity of BLI was nevertheless sufficient to monitor bioluminescence signals generated from the mouse IL-PFC during stress adaptation. Furthermore, the stereotactic procedure provides the added advantage of investigating neuroanatomical links between specific small areas of the prefrontal cortex such as a neurobiological interaction between the IL-PFC and prelimbic prefrontal cortex (PrL-PFC) in which GRs are expressed (Drevets et al., 2008; Myers-Schulz and Koenigs, 2012; van Aerde et al., 2008). The PrL-PFC is linked to the nucleus accumbens and basolateral amygdala, and plays a major role in control of stress response inhibition and reward. The IL-PFC is connected to visceral/emotional effector systems (i.e. the central amygdaloid nucleus and hindbrain cardiovascular regulatory pathways) and is important for the control of emotional responses to fear as well as activation of stress effector pathways (Vertes, 2004).

In conclusion, to our knowledge, this is the first report demonstrating the monitoring of GR signals during the entire stress adaptation process in the mouse IL-PFC. Employing our methodology as a platform to study GR activity might open new possibilities to elucidate the spatio-temporal dimensions of the molecular changes occurring upon GR activation during the process of stress-related mental disorders, such as post-traumatic stress disorder and depression. We also anticipate using this system to address additional pharmacological and physiological challenges, and to test the utility of this platform for monitoring other dynamic transcription factor activities in organs other than the brain.

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