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    • Here we used a designer receptors

    2022-01-20

    Here, we used a designer receptors activated exclusively by designer drugs (DREADD)-based chemogenetic approach in a novel Ghsr-IRES-Cre knock-in mouse line to test whether the activity of MBH GHSR neurons is required for the normal rebound food intake following fasting. We also used the Ghsr-IRES-Cre line to map the projections of the MBH GHSR neurons and confirm the central expression pattern of GHSR. We believe that the novel Ghsr-IRES-Cre mouse line described herein will further aid in the identification of the neurocircuitry directly engaged by ghrelin and the interaction of this ghrelin-responsive neurocircuitry with other factors and inputs that regulate food intake and body weight.
    Material and methods
    Results
    Discussion In this study, we describe a novel Ghsr-IRES-Cre knock-in mouse line that has allowed the molecular manipulation of ORY-1001 synthesis characterized by their expression of GHSR. Here, we focused on GHSR-expressing neurons, first investigating their central distribution. In particular, neuroanatomical characterization of Cre-dependent reporter expression in Ghsr-IRES-Cre mice crossed to either ROSA26-YFP or ROSA26-ZsGreen mice revealed a pattern of Cre activity that was largely consistent with the GHSR expression pattern observed previously using a combination of ISHH [19], [20], [23] plus a GHSR-eGFP transgenic reporter mouse line [23] and confirmed using other methods. Although those previous methods have been meritorious, the patterns of GHSR expression they reveal underrepresent the full extent of GHSR expression in the brain. For instance, GHSR mRNA expression in the dorsomedial and central aspects of the VMH as assessed by ISHH is noticeably absent in the mouse using ISHH and is sparse in the GHSR-eGFP reporter, whereas it had been expected to be marked based on ISHH findings in the rat [20], [23]. Similarly, while Arc expression of GHSR is marked by ISHH in the mouse, it is sparse in the GHSR-eGFP reporter [20], [23]. More broadly, ISHH seems to under represent GHSR expression in the cortex, hippocampus, VMH, and amygdala, whereas the GHSR-eGFP reporter underrepresents GHSR expression within the MBH and the midbrain [20], [23]. As described here, the pattern of Cre activity in the Ghsr-IRES-Cre line encompasses the regions missed or underrepresented by either ISHH or the GHSR-eGFP model alone. The Ghsr-IRES-Cre line also demonstrates Cre activity within the BNST and LSD, neither of which had been reported with ISHH or the GHSR-eGFP model [20], [23]. The BNST and LSD are important neural centers that receive and integrate inputs from the limbic system and are involved in the regulation of metabolism, mood, motivation, and behavioral stress responses [59], [60], [61], [62]. Many of these responses are also influenced by GHSR action [6], [63], [64], and future studies should assess whether GHSRs expressed within the BNST and/or LSD are involved in mediating those responses. Thus, the novel Ghsr-IRES-Cre line sensitively reports the central expression pattern of GHSR and as such should serve as an effective tool to investigate the chemical and electrophysiological properties and neurocircuitry of ghrelin-responsive, GHSR-expressing neurons, as we have begun here. A possible caveat in using the Ghsr-IRES-Cre driver line to manipulate GHSR-expressing cells derives from the potential for temporal variance in GHSR expression, and thus Cre activity, during development vs. adult life and for differential stability of GHSR and Cre. Thus, a scenario could exist in which strong, transient GHSR expression in the fetal period within a particular region could lead to strong Cre expression-induced changes in that region that persist into adulthood. Such temporal variance could influence the fidelity of the Ghsr-IRES-Cre driver line, potentially contributing to some of the observed differences with the GHSR expression pattern as revealed by ISHH. That said, the overall similarity of the GHSR expression pattern in the Ghsr-IRES-Cre line to the expression pattern as determined, for instance using ISHH, suggests that any such influence is likely not widespread.