Optogenetic manipulation of neuroendocrine rhythms – Opto-Rhythms

Development of a Cre-Lox strategy in order to control cell activities using selective opson expression
To do so, Cre (Dat or GHRH) mice were crossed with R26 fl/fl ChR2 mice and pulses of blue laser light triggered spiking activities up to 25Hz in patch-clamp recordings of neurons. In vivo experiments in which TIDA/GHRH neurons are (blue) light-activated (200 µm fiberoptic implanted in the third ventricule near the median eminence) whilst mPRL/mGH is monitored using a highly sensitive mPRL/mGH ELISA (1-4µl/blood sample collected from a tail vein) in freely-moving mice

1/ We generated a new transgenic mouse line, GHRH-Cre, which now allows us investigating which electric patterns in these parvocellular neurons control the build-up of hormone pulses.
2/ We developed a new, highly sensitive ELISA for PRL detection in very small volumes of mouse blood (1-2 µl).
3/ We succeeded in controlling blood flow rate in pituitary capillaries by blue laser-light activation of ChR2 selectively expressed in pericytes (Wnt1Cre x R26 fl/fl ChR2).

Using our new mouse models and techniques/tools, we will now explore:
1/ which are electrical patterns of GHRH/TIDA neurons effectively controlling hormone outputs from the pituitary gland
2/ the role of pituitary pericytes in the build-up of hormone pulses

Preliminary experiments were prossissing. We expect to prepare two papers for submission to high profile journals in a near future

In higher organisms, the co-ordinated secretion of pituitary hormones is essential for regulating basic body functions such as growth, reproduction and lactation. Pituitary secretions are primarily under the control of hypothalamic hypophysiotropic neurons, which release their signalling factors into the portal blood vessels at the level of the median eminence. Defaults in hormone levels and their rhythms are signatures of many hormonal disorders which are major and economically costly health care problems (dwarfism, infertility, metabolic disorders…). However, little is known about the in vivo rhythms of hypophysiotropic neuron activity that drive pituitary hormone responses. Similarly, the pituitary gland is still an enigma regarding how endocrine cells receive and respond to blood-borne hypothalamic stimuli, as well as how hormone outputs are finally delivered to the complex network of pituitary capillaries.

During the former Pit-Net grant, we (Mtp and London groups) have changed the view of how both GH- and PRL-secreting cells are functionally organized within the pituitary. In the past, this gland was considered to be a mosaic of distinct endocrine cell types so that functional assessment of these apparently dispersed cells was traditionally based on cell numbers and activities. Using pituitary-scale 2-photon imaging and functional in situ assays (Mtp groups) on tractable transgenic mice tagged with fluorescent proteins (GH-eGFP, PRL-DsRed…) (London group and other collaborators), we pioneered integrative developmental, morphological and functional studies showing that the pituitary gland is composed of interacting cell networks (PNAS 2005, J. Endoc. 2009, Endocrinology 2010). Another fascinating outcome of the Pit-Net project has been our recent development of in vivo approaches to measure local blood flow, oxygen partial pressure and cell activity at single-cell resolution in mouse pituitary glands in situ. These methods involved modifying a fluorescent stereomicroscope with long working distance objectives to image an exposed pituitary gland deep in its in vivo environment at both wide field and single cell resolution (Lafont et al. PNAS, final revision; see also ipam.igf.cnrs.fr/).

Cellular in vivo imaging and other functional assays will now be used to fill the critical gaps in our understanding of how the pituitary cell networks receive and decode their native hypothalamic inputs. Two systems will be studied: 1) GH-cells which respond to GHRH by forming pulses of hormone; and 2) lactotrophs which are negatively regulated by dopamine (released by TIDA neurons) due to their high basal activity. Mollard’s group proposes to continue collaborating with Paul Le Tissier's group as well as beginning a new collaboration with Ulrich Boehm (Center for Molecular Neurobiology, Hamburg). Boehm’s group has recently generated ROSA26-driven mice floxed for light-activatable opsin-based tools, either the depolarizing blue light-gated cation channel channelrhodopsin-2 [ChR2] or the hyperpolarizing yellow light-driven chloride pump halorhodopsin [NpHR]. The ChR2 and NpHR are tagged with the fluorescent proteins YFP and mCherry, respectively, to allow identification of their specific cellular location. Boehm’s group has already validated the R26-NpHR-mCherry mice which were successfully crossed with neurohormone-Cre animals, allowing the hormonal manipulation. R26-ChR2-YFP mice are currently under testing. Based on the synergistic and complementary expertises of the three groups forming the proposed ANR team, we are confident that we can achieve the following goals: 1) to determine how the pattern of hypothalamic stimulation influences pituitary gland output; 2) precisely identify how the different topological arrangements of endocrine cell networks in the pituitary gland determines specific functions; and 3) to characterize the role of the relationship between the microcirculation and endocrine cell networks.