Division of Hypothalamic Research, Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, TX, USA.
Monash Biomedicine Discovery Institute and Department of Physiology, Faculty of Medicine, Monash University, Clayton, Victoria, Australia.
Mol Metab. 2017 Jun 22;6(8):882-896. doi: 10.1016/j.molmet.2017.06.011. eCollection 2017 Aug.
Ghrelin is a stomach-derived hormone that affects food intake and regulates blood glucose. The best-characterized actions of ghrelin are mediated by its binding to and activation of the growth hormone secretagogue receptor (GHSR; ghrelin receptor). Adequate examination of the identity, function, and relevance of specific subsets of GHSR-expressing neurons has been hampered by the absence of a suitable Cre recombinase (Cre)-expressing mouse line with which to manipulate gene expression in a targeted fashion within GHSR-expressing neurons. The present study aims to characterize the functional significance and neurocircuitry of GHSR-expressing neurons in the mediobasal hypothalamus (MBH), as they relate to ghrelin-induced food intake and fasting-associated rebound hyperphagia, using a novel mouse line in which Cre expression is controlled by the promoter.
A mouse line that expresses Cre directed by the promoter was generated. The line was validated by comparing Cre activity in reporter mice to the known brain distribution pattern of GHSR. Next, the requirement of MBH GHSR-expressing neuronal activity in mediating food intake in response to administered ghrelin and in response to fasting was assessed after stereotaxic delivery of inhibitory designer receptor exclusively activated by designer drugs (DREADD) virus to the MBH. In a separate cohort of mice, stereotaxic delivery of stimulatory DREADD virus to the MBH was performed to assess the sufficiency of MBH GHSR-expressing neuronal activity on food intake. Finally, the distribution of MBH GHSR-expressing neuronal axonal projections was assessed in the DREADD virus-injected animals.
The pattern of Cre activity in the mouse line mostly faithfully reproduced the known GHSR expression pattern. DREADD-assisted inhibition of MBH GHSR neuronal activity robustly suppressed the normal orexigenic response to ghrelin and fasting-associated rebound food intake. DREADD-assisted stimulation of MBH GHSR neuronal activity was sufficient to induce food intake. Axonal projections of GHSR-expressing MBH neurons were observed in a subset of hypothalamic and extra-hypothalamic regions.
These results suggest that 1) activation of GHSR-expressing neurons in the MBH is required for the normal feeding responses following both peripheral administration of ghrelin and fasting, 2) activation of MBH GHSR-expressing neurons is sufficient to induce feeding, and 3) axonal projections to a subset of hypothalamic and/or extra-hypothalamic regions likely mediate these responses. The line should serve as a valuable tool to further our understanding of the functional significance of ghrelin-responsive/GHSR-expressing neurons and the neuronal circuitry within which they act.
Ghrelin 是一种源自胃的激素，它影响食物摄入并调节血糖。Ghrelin 的最佳特征作用是通过其与生长激素促分泌受体（GHSR；ghrelin 受体）结合并激活来介导的。由于缺乏合适的 Cre 重组酶（Cre）表达小鼠系，因此无法以靶向方式操纵 GHSR 表达神经元中的基因表达，从而充分检查 GHSR 表达神经元的身份、功能和相关性特定亚群。本研究旨在使用新型小鼠系来研究中脑基底部（MBH）中 GHSR 表达神经元的功能意义和神经回路，这些神经元与 ghrelin 诱导的食物摄入和禁食相关的反弹性暴食有关，该小鼠系中 Cre 表达受 启动子控制。
生成了一种表达 Cre 的新型小鼠系，该 Cre 由 启动子驱动。通过将报告小鼠中的 Cre 活性与 GHSR 的已知脑分布模式进行比较，对该系进行了验证。接下来，在立体定向递送抑制性 Designer 受体专门激活 Designer 药物（DREADD）病毒到 MBH 后，评估了 MBH GHSR 表达神经元活性在响应给予的 ghrelin 和响应禁食时介导食物摄入的必要性。在 小鼠的另一队列中，通过立体定向递送兴奋性 DREADD 病毒到 MBH，评估了 MBH GHSR 表达神经元活性对食物摄入的充分性。最后，在接受 DREADD 病毒注射的动物中评估了 MBH GHSR 表达神经元轴突投射的分布。
该小鼠系中的 Cre 活性模式大多忠实地再现了已知的 GHSR 表达模式。DREADD 辅助抑制 MBH GHSR 神经元活性强烈抑制了 ghrelin 外周给药和禁食相关的反弹性食物摄入的正常食欲作用。MBH GHSR 表达神经元的 DREADD 辅助刺激足以诱导进食。GHSR 表达 MBH 神经元的轴突投射在下丘脑和下丘脑外的一些区域被观察到。
这些结果表明，1）外周给予 ghrelin 和禁食后，MBH 中 GHSR 表达神经元的激活是正常进食反应所必需的，2）MBH GHSR 表达神经元的激活足以诱导进食，3）到一组下丘脑和/或下丘脑外区域的轴突投射可能介导这些反应。该小鼠系应成为进一步了解 ghrelin 反应性/GHSR 表达神经元及其作用的神经元回路的功能意义的有价值的工具。
