Scene of the crime: where is GnRH released, and what might this tell us?

“Not where, when.” Gaudior to Charles Wallace in A Swiftly Tilting Planet by Madeline L'Engle

For much of the four-decade history of the study of GnRH neurons, investigators have seemingly heeded Gaudior's advice, and the study of when has mattered more than that of where. This predominance has stemmed from elegant demonstrations of the critical nature of the pattern of GnRH release for normal fertility (1). An intriguing new study in this issue of Endocrinology by Fuenzalida et al. (2) suggests attention should also be focused on the question of where GnRH release occurs.

These investigators used an established culture system for embryonic primate GnRH neurons (3) in combination with a membrane-impermeant dye, FM1-43, that is incorporated into and released from vesicles during endocytosis and exocytosis, respectively (4). Under basal conditions, FM1-43 was incorporated into vesicle-like structures both in the somata and in the processes of cultured GnRH neurons, suggesting spontaneous release of vesicles from both compartments. A strong depolarizing stimulus (56 mm K+) or veratridine, which activates sodium channels underlying action potential generation, both increased FM1-43 incorporation. The latter action was inhibited by tetrodotoxin, which blocks sodium channels. Interestingly, similar incorporation of FM1-43 was not observed in non-GnRH neurons from the same cultures or in cortical neurons cultured separately. This indicates somatic and neural process vesicle release is at least in part specialized to neurosecretory neurons such as GnRH neurons, although identification of the phenotype of the other neurons would be necessary to make a firm conclusion in this regard.

To further investigate the substances released and the nature of the release site, the investigators used a fixable form of the dye, FM1-43Fx. Subsequent double labeling revealed both GnRH decapeptide and the synaptic vesicle component vesicle-associated membrane protein within FM1-43Fx-positive fluorescent puncta, suggesting GnRH was among the substances being released. Finally, a second double-labeling study confirmed colocalization of GnRH and bassoon, a marker of the active neurosecretory zone. Together these studies suggest GnRH is released from somata and neural processes as well as from nerve terminals.

The present data add to a growing body of literature that indicate GnRH release is not just a terminal output from the brain to regulate the pituitary but also functions centrally. Such central effects were first suggested by the actions of GnRH to facilitate lordosis behavior in females (5) and by in vivo studies of autoregulation of GnRH neurons (6). More recent electrophysiological studies of GnRH action on GnRH neurons suggest the decapeptide can regulate these cells, although the direction of response remains debated and may indeed be a function of physiological state (7, 8). In addition, GnRH neurons have been proposed to regulate their GABAergic afferents through multiple mechanisms including GnRH release (9, 10). The notion of GnRH neuron-GnRH neuron communication via the GnRH decapeptide is a tantalizing one for synchronization of these widespread cells in a GnRH neuron-specific manner. With particular regard to the new finding of neurosecretion from the processes of these cells, a recent anatomical study revealed intricate associations among GnRH neuron dendrites, including specializations such as zonula adherens (11).

Together these observations indicate that future studies of GnRH neuronal function should examine where release is occurring in addition to when. Do the same stimuli induce release from terminals and other cell regions? Strong depolarization from a very high level of potassium does cause GnRH release from terminals in the median eminence (12) as well as dye uptake in processes as demonstrated by Fuenzalida et al. (2); however, endogenous depolarizing stimuli are likely to be milder. This raises interesting questions for future studies. For example, does GnRH release in vivo from dendrites and terminals occur at different times and in different patterns? Do terminal and dendritic release serve different functions? Are terminal and dendritic release differentially regulated by reproductive state and by different stimuli? Does maturation stage alter where on a GnRH neuron peptide is released? Are there species differences, and if so, where do these develop evolutionarily and what can that teach us? In the case in other mammalian neuroendocrine systems, both differential timing and function have been demonstrated, suggesting this may be the case for GnRH neurons as well (13).

As with any study, questions remain for future experiments. These observations were made in a culture system of immature GnRH neurons. Although these cultures have been well established to secrete GnRH neuropeptide and synchronize intracellular calcium oscillations at an interval consistent with pulsatile GnRH release in primates in vivo (14, 15), it is possible that the different organization of GnRH neurons in culture vs. in vivo could alter the results observed here. These technical questions notwithstanding, the present findings have provided what any successful study should: a plethora of additional questions.