Morphological assessment of GABA and glutamate inputs to GnRH neurons in intact female mice using expansion microscopy.

Shel‐Hwa Yeo,Allan E. Herbison,Michel K. Herde

doi:10.1111/jne.13021

Shel‐Hwa Yeo, Allan E. Herbison + Show 1 more

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https://doi.org/10.1111/jne.13021

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Abstract

The roles GABAergic and glutamatergic inputs in regulating the activity of the gonadotrophin-releasing hormone (GnRH) neurons at the time of the preovulatory surge remain unclear. We used expansion microscopy to compare the density of GABAergic and glutamatergic synapses on the GnRH neuron cell body and proximal dendrite in dioestrous and pro-oestrous female mice. An evaluation of all synapses immunoreactive for synaptophysin revealed that the highest density of inputs to rostral preoptic area GnRH neurons occurred within the first 45µm of the primary dendrite (approximately 0.19 synapsesµm-1 ) with relatively few synapses on the GnRH neuron soma or beyond 45µm of the dendrite (0.05-0.08 synapsesµm-1 ). Triple immunofluorescence labelling demonstrated a predominance of glutamatergic signalling with twice as many vesicular glutamate transporter 2 synapses detected compared to vesicular GABA transporter. Co-labelling with the GABAA receptor scaffold protein gephyrin and the glutamate receptor postsynaptic density marker Homer1 confirmed these observations, as well as the different spatial distribution of GABA and glutamate inputs along the dendrite. Quantitative assessments revealed no differences in synaptophysin, GABA or glutamate synapses at the proximal dendrite and soma of GnRH neurons between dioestrous and pro-oestrous mice. Taken together, these studies demonstrate that the GnRH neuron receives twice as many glutamatergic synapses compared to GABAergic synapses and that these inputs preferentially target the first 45µm of the GnRH neuron proximal dendrite. These inputs appear to be structurally stable before the onset of pro-oestrous GnRH surge.

Highlights

The gonadotrophin-releasing hormone (GnRH) neurons represent the final output cells of the neural network controlling fertility in mammals
There is little doubt that sustained high levels of circulating oestradiol are obligatory for the GnRH surge in spontaneously ovulating mammals[5] and this is relayed to the GnRH neurons by afferent inputs that express the key oestrogen receptor, ESR1.6-8 These neurons very likely target the GnRH neuron cell body and proximal dendrites to trigger the intense neuronal activation required for the surge.[9,10]
Electrophysiological studies have reported that GABAA and glutamate receptor transmission at the GnRH neuron can change at the time of the luteinising hormone (LH) surge,[15,16] this depends on the animal model used.[17,18]

Summary

| INTRODUCTION

The gonadotrophin-releasing hormone (GnRH) neurons represent the final output cells of the neural network controlling fertility in mammals. How the GnRH neurons become suddenly and intensely activated to create the massive outpouring of GnRH that occurs at the surge remains unclear.[3,4] There is little doubt that sustained high levels of circulating oestradiol are obligatory for the GnRH surge in spontaneously ovulating mammals[5] and this is relayed to the GnRH neurons by afferent inputs that express the key oestrogen receptor, ESR1.6-8 These neurons very likely target the GnRH neuron cell body and proximal dendrites to trigger the intense neuronal activation required for the surge.[9,10]. Moore et al[21] recently examined this issue in an ovariectomised, oestradiol-treated mouse model and, surprisingly, found little change in the numbers of vesicular GABA transporter (VGAT) or VGLUT2-immunoreactive terminals opposing the GnRH neuron cell body and proximal dendrites around the time of the LH surge. Expansion microscopy (ExM) utilises isotropic tissue swelling to expand the sample so that regular confocal imaging can achieve the necessary resolution required to identify bona fide synapses in the brain.[22]

| MATERIALS AND METHODS

Findings

| DISCUSSION