Abstract
The ability to pass on genetic information through reproduction represents nature’s basic element of existence. Central to the control of reproduction is gonadotropin-releasing hormone (GnRH), a neuropeptide synthesized and released from GnRH neurons in the mammalian hypothalamus. Our understanding of these neurons has traditionally been limited to examination of the cell body and very proximal dendrite. However, recent technological advances have revealed that GnRH neurons possess extensive dendrites with numerous morphological features that aid in our understanding of their physiology. Despite this, current research has been limited to in vitro brain slice preparations that do not always encompass the entire extent of GnRH neurons and limit our ability to investigate specific in vivo physiological time points.
In light of this, the first aim of the present study was to image the morphology of whole GnRH neurons in optically cleared, adenoviral injected mouse brains. We optimized a technical approach for studying GnRH neurons that combines in vivo cell-filling of GnRH neurons with optical tissue clearing and long working distance confocal microscopy. This novel approach utilized adenoviral vector-mediated expression of farnesylated enhanced green fluorescent protein (Ad-iZ/EGFPf) to “fill” whole GnRH neurons in vivo via a cranial injection. Following Ad-iZ/EGFPf injection, perfusion fixed mouse brains were made transparent by immersion in Scaleview-A2, a chemical solution that reduces the light absorbance of tissue, enabling imaging of deeper structures in thick tissue samples. Tissue absorbance was reduced from an average of 2.060 ± 0.09au in uncleared samples to 0.4914 ± 0.08au in Scaleview-A2 treated samples, at a wavelength of 490nm. In addition, we found injections of Ad-iZ/EGFPf to the rostral pre-optic area resulted in the expression of EGFPf along the entire cell membrane of a small population of GnRH neurons. After brains had been cleared for 14 days, long working distance confocal microscopy enabled visualisation of somal, dendritic and axonal compartments of GnRH neurons that could be traced on a millimetre scale, a feat that has challenged scientists in recent years. We were also able to identify microscopic features of GnRH neurons at a depth of >400μm through the brain tissue.
The second objective of this study was to investigate the changes in GnRH neuron morphology associated with neuronal activation at the pre-ovulatory surge, a physiologically critical time point in females. The compatibility of chemical clearing with immunohistochemistry was confirmed with staining for c-Fos, a marker of neuronal activation. The LH plasma concentration from estradiol benzoate treated (n=5) and vehicle treated animals (n=5) was analysed to confirm the induction of the pre-ovulatory surge, before 1.5mm thick sagittal sections were immunostained for c-Fos. Tissue samples displayed c-Fos staining to an average depth of 130μm, however, some filled GnRH neurons were found at 400μm. Consequently no c-Fos positive GnRH neurons were identified meaning they could not be explicitly categorized as either activated or non-activated. Despite our inability to investigate changes in morphological features of GnRH neurons at this time point, unique features of the GnRH neurons identified with this novel technique were explored.
Overall, the establishment of in vivo cell-filling in large volume tissue samples, and the subsequent ability to visualise and image entire GnRH neurons represents a great leap in GnRH research, taking us beyond the soma and proximal dendrite, and beyond in vitro brain slice preparations.