Different dendritic domains of the GnRH neuron underlie the pulse and surge modes of GnRH secretion in female mice
- Department of Assisted Reproduction, Shanghai Ninth People’s Hospital, Shanghai JiaoTong University School of Medicine, China
- Centre for Neuroendocrinology, Department of Physiology, University of Otago, New Zealand
- Department of Physiology, Development and Neuroscience, University of Cambridge, United Kingdom
- School of Basic Medical Sciences, Institutes of Brain Science, Fudan University, China
Figures
Figure 1 with 1 supplement
Expansion microscopy views of synaptic appositions at the GnRH neuron proximal dendrite and distal dendron.
Schematic showing the morphology of a hypophysiotropic GnRH neuron with its soma-proximal dendrites located in the rostral preoptic area (rPOA) and the distal dendron and short axon branches in the …
Figure 1—figure supplement 1
Definition of GABAA receptor synapses on GnRH neurons using ExM.
(A) Example of a ‘side-on’ view of a GABAergic synapse on a GnRH proximal dendrite. Insert, magnified image from boxed region showing the plane of analysis used to generate the fluorescence …
Figure 2 with 1 supplement
Chemogenetic inhibition of GnRH neurons by intraperitoneal (iP) injection of CNO suppresses both the surge and pulse profiles of LH secretion.
(A) Schematic showing experimental protocol with GnRH-Cre mice injected with Cre-dependent Flex-hM4D(Gi)-mCherry AAVs bilaterally into the region of the median eminence and CNO given by IP …
Figure 2—figure supplement 1
LH surge profiles of wild-type and un-injected GnRH-Cre mice and expression of hM4D(Gi)-mCherry in GnRH neurons.
LH surge profiles in all individual wild-type (A, n = 7) and GnRH-cre (B, n = 8) OVX+E+P female mice over 6 hr, with tail-tip blood sampling every hour. (C) Mean (± SEM) LH levels in wild-type and …
Figure 3 with 1 supplement
Chemogenetic inhibition of GnRH neuron distal dendron activity suppresses both the surge and pulse profiles of LH secretion.
(A) Schematic showing experimental protocol with GnRH-Cre mice injected with Cre-dependent Flex-hM4D(Gi)-mCherry AAVs bilaterally into the region of the median eminence and CNO given into the same …
Figure 3—figure supplement 1
Photomicrographs showing location of cannula and mean (± SEM) LH levels in GnRH-Cre OVX mice with saline and CNO.
(A) Photomicrograph showing location of cannula implanted into the MBH of GnRH::Flex-hM4D(Gi)-mCherry mice. Scale bar, 100 μm. Mean (± SEM) LH levels in AAV-injected GnRH-Cre OVX mice with IP …
Figure 4
Chemogenetic inhibition of GnRH neuron soma-proximal dendrite activity suppresses only the LH surge.
(A) Schematic showing experimental protocol in which GnRH-Cre mice are injected with Cre-dependent Flex-hM4D(Gi)-mCherry AAVs bilaterally into the region of the median eminence and, later, CNO …
Figure 5 with 2 supplements
Bilateral optogenetic inhibition of GnRH neuron soma-proximal dendrite activity suppresses only the LH surge.
(A) Schematic showing experimental protocol with GnRH-cre+/-;Rosa-CAG-Arch-GFP+/+ mice with bilateral optic fiber placement in the rostral preoptic area (rPOA). (B) Fluorescence images of GnRH …
Figure 5—figure supplement 1
Archaerhodopsin expression in GnRH neurons and effects of yellow light on GnRH neuron firing.
(A) Photomicrographs showing Archaerhodopsin (GFP reporter), GnRH (red), and overlay in the GnRH-cre+/-;Rosa-CAG-Arch3-GFP+/+ mice. Scale bar, 50 µm. Whole cell current clamp recordings of …
Figure 5—figure supplement 2
LH surge in GnRH-Arch mice and optogenetic control experiments showing effects of yellow light on the LH surge and pulses in wild-type mice.
(A) LH surge profiles in individual GnRH-Cre+/-;Rosa-CAG-Arch-GFP+/+ OVX+E+P female mice over 6 hr, with tail-tip blood sampling every hour. (B) Mean (± SEM) LH surge profile. Values significantly …
Author response image 1
Cell-attached recording of a GnRH neuron from an AAV-injected mouse showing the effect of 1 μM CNO on firing.
Tables
Table 1
Quantitative analysis of synaptic appositions on the proximal and distal dendrites of GnRH neurons in diestrus female mice (N = 4).
For proximal dendrites, 60 µm of dendrite was measure in each case, whereas 15 µm lengths were examined for distal dendrites.
| GnRH proximal dendrites | |
|---|---|
| No. of dendrites counted | 38 |
| Percentage of dendrites with synaptophysin contact | 95% |
| No. of synaptophysin contacts with GnRH dendrite | 183 |
| Synaptic density per 10 µm | 0.8 ± 0.3 |
| GnRH Distal Dendrites | |
| No. of dendrites counted | 211 |
| Percentage of dendrites with synaptophysin contact | 53% |
| No. of synaptophysin contacts with GnRH dendrite | 330 |
| Synaptic density per 10 µm | 2.0 ± 0.2 |
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Genetic reagent (M. musculus) | STOCK Tg(Gnrh1-cre)1Dlc/J | Jackson Laboratory | Stock #: 021207 RRID:IMSR_JAX:021207 | |
| Genetic reagent (M. musculus) | B6;129S-Gt(ROSA)26Sortm35.1(CAG-AOP3/GFP)Hze/J | Jackson Laboratory | Stock #: 012735 RRID:IMSR_JAX:012735 | |
| Genetic reagent (M. musculus) | B6.DBA-Tg(Gnrh1-EGFP)1Phs | Spergel et al., 1999 | MGI:6158458 | |
| Transfected construct (M. musculus) | AAV2-Retro-hSyn-DIO-hM4D(Gi)-mCherry | OBiO Technology (Shanghai) Corp., Ltd | RRID:Addgene_44362 | |
| Antibody | polyclonal guinea pig anti-GnRH antisera | gift G. Anderson, University of Otago | Cat #GA02 RRID:AB_2721118 | (1: 5000) |
| Antibody | polyclonal rabbit anti-GnRH antisera | gift G. Anderson, University of Otago | Cat #GA01 RRID:AB_2721114 | (1: 5000) |
| Antibody | Alexa Fluor 488-AffiniPure Donkey Anti-Guinea Pig IgG (H+L) | Jackson ImmunoResearch Labs | Cat# 706-545-148, RRID:AB_2340472 | (1:500) |
| Antibody | Alexa Fluor 594 donkey anti-rabbit antibody | Jackson ImmunoResearch Labs | Cat# 711-585-152, RRID:AB_2340621 | (1:500) |
| Antibody | DAPI (4',6-Diamidino-2-Phenylindole, Dilactate) antibody | Thermo Fisher Scientific | Cat# D3571, RRID:AB_2307445 | 300 nM |
| Antibody | Anti-GFP (chicken polyclonal) | Abcam | Cat# AB13970 RRID:AB_300798 | (1:8000) |
| Antibody | Anti-Synaptophysin 1 (guinea pig polyclonal) | Synaptic Systems | Cat#101004 RRID:AB_1210382 | (1:800) |
| Antibody | Anti-Vesicular GABA Transporter (rabbit polyclonal) | Synaptic Systems | Cat#131003 RRID:AB_887869 | (1:800) |
| Antibody | Anti- Gephyrin (guinea pig polyclonal) | Synaptic Systems | Cat#147318 RRID:AB_2661777 | (1:800) |
| Antibody | Goat anti-chicken (goat polyclonal, Alexa488-conjugate) | Thermo Fisher Scientific | Cat# A-11039 RRID:AB_2534096 | (1:200) |
| Antibody | Goat anti-guinea pig (goat polyclonal, biotin-conjugated) | Vector Laboratories | Cat# BA-7000 RRID:AB_2336132 | (1:200) |
| Antibody | Goat anti-rabbit (goat polyclonal, ATTO647N-conjugated) | Sigma-Aldrich | Cat# 40839 RRID:AB_1137669 | (1:200) |
| Chemical compound, drug | Beta-Estradiol 3-benzoate | Sigma Aldrich | E8515 | |
| Chemical compound, drug | Beta estradiol | Sigma Aldrich | E8875 | |
| Chemical compound, drug | progesterone | Sigma Aldrich | P0130 | |
| Chemical compound, drug | clozapine-N- oxide | Sigma Aldrich | C0832 | |
| Software, algorithm | GraphPad Prism software | GraphPad Prism | RRID:SCR_002798 | Version 6.01 |
| Software, algorithm | ImageJ image analysis software | ImageJ (https://imagej.net/) | RRID:SCR_003070 | |
| Software, algorithm | Vaa3D data visualization software | Vaa3D (http://www.vaa3d.org) | RRID:SCR_002609 |
