GnRH Neuron Firing and Response to GABA in Vitro Depend on Acute Brain Slice Thickness and Orientation

Abstract

The GnRH neurons exhibit long dendrites and project to the median eminence. The aim of the present study was to generate an acute brain slice preparation that enabled recordings to be undertaken from GnRH neurons maintaining the full extent of their dendrites or axons. A thick, horizontal brain slice was developed, in which it was possible to record from the horizontally oriented GnRH neurons located in the anterior hypothalamic area (AHA). In vivo studies showed that the majority of AHA GnRH neurons projected outside the blood-brain barrier and expressed c-Fos at the time of the GnRH surge. On-cell recordings compared AHA GnRH neurons in the horizontal slice (AHAh) with AHA and preoptic area (POA) GnRH neurons in coronal slices [POA coronal (POAc) and AHA coronal (AHAc), respectively]. AHAh GnRH neurons exhibited tighter burst firing compared with other slice orientations. Although α-Amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) excited GnRH neurons in all preparations, γ-aminobutyric acid (GABA) was excitatory in AHAc and POAc but inhibitory in AHAh slices. GABA(A) receptor postsynaptic currents were the same in AHAh and AHAc slices. Intriguingly, direct activation of GABA(A) or GABA(B) receptors respectively stimulated and inhibited GnRH neurons regardless of slice orientation. Subsequent experiments indicated that net GABA effects were determined by differences in the ratio of GABA(A) and GABA(B) receptor-mediated effects in "long" and "short" dendrites of GnRH neurons in the different slice orientations. These studies document a new brain slice preparation for recording from GnRH neurons with their extensive dendrites/axons and highlight the importance of GnRH neuron orientation relative to the angle of brain slicing in studying these neurons in vitro.

Differential Regulation of GnRH Secretion in the Preoptic Area (POA) and the Median Eminence (ME) in Male Mice

“…Because GnRH neurons express kiss1R, kisspeptin action is likely, at least in part, directly on GnRH neurons. Kisspeptin can, however, also excite GnRH neurons indirectly by increasing excitatory GABAergic as well as glutamatergic transmission to these cells (52,(55)(56)(57).…”

Progress and Challenges in the Search for the Mechanisms of Pulsatile Gonadotropin-Releasing Hormone Secretion

“…However, pulsatile GnRH/LH release requires serial sampling and even LH measurements are hardly achievable with mouse blood volume (124). At a cellular level, the first challenge is anatomical: preserving the connectivity with relevant inputs (125), GnRH cell morphology (126), and tracking a neuron within the complexity of the ME (20). The second challenge is technical as methods for detection of quantal secretion are not applicable to GnRH neurons: (1) synaptically coupled neurons are recorded simultaneously in brain slices [reviewed in Ref.…”

“…(40, 132, 133)]}. Although most GnRH neurons display autonomous firing of APs, firing is heterogeneous among GnRH neurons (126), far from an oscillatory activity that could trigger pulses every ~20 min (124). Even in vivo GnRH neurons exhibit heterogeneous behavior (114).…”

Morphological Characterization of the Action Potential Initiation Segment in GnRH Neuron Dendrites and Axons of Male Mice

“…Briefly, 200-m-thick coronal (for GnRH neurons around OVLT), sagittal (for MS and rPOA), or 400-m-thick ventral horizontal brain slices (for AHA/ME) were prepared (21,22) and GnRH neurons identified under fluorescence and a tight seal configuration (0.05-2 G⍀) was obtained using patch pipettes of 8 -12 M⍀ resistance (G150TF-3; Warner Instruments, Hamden, CT). The pipette solution (299 mosmol/kg, pH 7.3) contained (in mM) 135 K-gluconate, 5 NaCl, 10 HEPES, 10 EGTA, 1 MgCl 2 , 1 CaCl 2 , 5 MgATP, and 0.1 Na 2 GTP and 0.2% neurobiotin (SP-1120; Vector Laboratories, Burlingame, CA).…”