Figure 1. 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 median eminence (ME). Synaptic density analysis was undertaken on 60 μm-lengths of proximal dendrite and 15 μm-lengths of distal dendron. (A) Expansion microscopy view of a proximal dendrite (green) with surrounding synaptophysin puncta (red). (B) shows rotated 3D reconstruction with white lines indicating three appositions that were examined. (C) The side-on relative fluorescence intensity profiles are shown for the three appositions. (i) and (iii) represent synaptic appositions whereas (ii) indicates apposing profiles with no overlap that do not represent a synapse. (D) Expansion microscopy view of distal dendrons (green) with surrounding red synaptophysin puncta. (E) shows rotated 3D reconstruction with white lines indicating three appositions that were examined. (F) The relative fluorescence intensity profiles are shown for the three appositions. (ii) and (iii) represent synaptic appositions, whereas (i) indicates apposing profiles with no overlap that do not represent a synapse. Scale bars show pre-expansion values with expanded size in brackets. X-axis plots show pre- expansion values.

Figure 1—figure supplement 1. Definition of GABA_A 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 intensity profiles of each channel - GFP (GnRH proximal dendrites; green), Alexa568 (gephyrin; red) and ATTO647 (VGAT, blue). Analysis of intensity profiles from 25 ‘side-on’ synapses demonstrated that the overlap in signals between the pre-synaptic marker and GnRH neuron was always >0.95 µm (0.23 µm pre-expansion). (B) Example of a ‘face-on’ view of a GABAergic synapse on a GnRH proximal dendrite. Insert, magnified image from boxed region showing the z-plane of analysis used to generate the fluorescence intensity profiles of each channel - GFP (GnRH proximal dendrites; green), Alexa568 (gephyrin; red) and ATTO647 (VGAT, blue). Analysis of intensity profiles from 12 ‘face-on’ synapses demonstrated that the z-axis overlap in signals between the pre-synaptic marker VGAT and GFP of the GnRH neuron was >1.75 µm (0.42 µm pre-expansion) for all synapses.