In situ GABAergic modulation of synchronous gonadotropin releasing hormone-1 neuronal activity

Abstract

Evidence indicates that gonadotropin releasing hormone-1 [GnRH-1, also known as luteinizing hormone releasing hormone (LHRH)] neurons can exhibit synchronized neuroendocrine secretory activity before entrance into the CNS. In this study, we used calcium imaging to evaluate patterns of activity in individual, embryonic, GnRH-1 neurons as well as population dynamics of GnRH-1 neurons in mouse nasal explants maintained for 1 versus 3 weeks. Independent of age, GnRH-1 neurons displayed significant calcium peaks that synchronized at an interval of approximately 20 min across multiple GnRH-1 cells within an explant. Acute tetrodotoxin treatment decreased the amplitude of calcium peaks in individual GnRH-1 neurons and the duration but not the frequency of synchronized activity in the population of GnRH-1 neurons. Acute GABA(B) receptor antagonism increased the frequency of synchronized neuronal activity at both ages, whereas acute GABA(A) receptor antagonism decreased calcium oscillations in individual GNRH-1 cells as well as synchronization of the calcium pulses within the GnRH-1 population at the 1 week time point to background non-GNRH-1 cell levels. These results indicate that developing GnRH-1 neurons rely heavily on GABAergic signaling to initiate synchronized bouts of activity but thereafter, possess an innate capacity for synchronized activity patterns that are modulated by, but not completely dependent on GABAergic signaling.

Fig. 1.

Changes in intracellular calcium can be used to evaluate activity patterns in GnRH-1 neurons. A–D, Digitized images of neurons in nasal explants. Calcium imaging (A, C) and GnRH-1 immunofluorescence (B, D) for explants at 1 week (A, B) and 3 weeks (C, D) in vitro. Neurons in the periphery that label with Calcium Green are predominantly GnRH-1 neurons (compare A, B; C, D).Arrows indicate clusters of GnRH-1 neurons, whereassmall arrowheads indicate single GnRH-1 neurons.Oversized arrowheads (A) indicate non-GnRH-1 cells loaded with Calcium Green. E, Calcium Green trace of SFM-treated culture quantified from digital images obtained at 5 sec intervals. Top graph shows complete recording over 22 min. Hatch marks at the topof the graphs in E demonstrate significant peaks as detected by the PULSAR algorithm. Bottom graphs inE (expanded data from boxed areas intop graph) demonstrate duration of significant peaks.F, Calcium Green traces in two individual cells from the same SFM-treated explant displaying unique activity patterns.

Fig. 2.

Calcium peaks are rarely observed in non-GnRH-1 cells in 1-week-old (A, B) and 3-week-old (C, D) nasal explants. Calcium traces from individual non-GnRH-1 cells maintained in SFM. Values represent mean optical density, after background correction, of Calcium Green-1 fluorescence within the cell soma.

Fig. 3.

Calcium fluctuations in GnRH-1 neurons in 1-week-old nasal explants. Calcium traces from individual GnRH-1 neurons treated acutely with serum-free media (A, SFM1), tetrodotoxin (B, TTX1), picrotoxin (C, PIC1), or saclofen (D, SAC1). Values represent mean optical density, after background correction, of Calcium Green-1 fluorescence within the cell soma. Asterisks indicate significant peaks in intracellular calcium as determined by PULSAR analysis.

Fig. 4.

Calcium fluctuations in GnRH-1 neurons in 3-week-old nasal explants. Calcium traces from individual GnRH-1 neurons treated acutely with serum-free media (A, SFM3), tetrodotoxin (B, TTX3), picrotoxin (C, PIC3), or saclofen (D, SAC3). Values represent mean optical density, after background correction, of Calcium Green-1 fluorescence within the cell soma. Asterisks indicate significant peaks in intracellular calcium as determined by PULSAR analysis.

Fig. 5.

Synchronous neuronal activity is observed in GnRH-1 neuronal populations. The top panel inA demonstrates WAVELET analysis (color contour plot) of a portion of the combined calcium activity of 10 cells (5 of which are shown below) from a single nasal explant preparation treated with SFM alone. Shown below (B, C) are the minute intervals (bottom box, tick marks) in which PULSAR detected calcium peaks and the WAVELET transform (top, color contour plots) for GnRH-1 neurons in a 1-week-old, SFM-treated explant (B, SFM1) and a 3-week-old, SFM-treated explant (C, SFM3). The bottom panels (withtick marks) indicate significant calcium peaks in 10 individual GnRH-1 neurons (rows) in a single explant preparation. The top panels in B andC (with color columns) represent the one-dimensional continuous sum of plotted cell activity (frombottom panel) in each preparation (see Materials and Methods). Asterisks in A indicate significant pulses of synchronized activity in the monitored population of GnRH-1 neurons. Stars on individual activity plots (A, bottom) indicate PULSAR detected peaks that occurred in WAVELET detected synchronized pulses; diamondsindicate PULSAR detected peaks only. Arrowheads inB indicate cells also shown in A. Independent of age, GnRH-1 neuronal populations exhibit synchronized calcium oscillations with an interval of ∼18 min.

Fig. 6.

Picrotoxin treatment alters GnRH-1 neuronal activity. Shown are the minute intervals (box, tick marks) in which PULSAR detected calcium peaks and the WAVELET transform (top, shaded contour plots) for a 1-week-old, picrotoxin-treated explant (A, PIC1) and a 3-week-old, picrotoxin-treated explant (B, PIC3). The bottom panels (with tick marks) indicate significant calcium peaks in nine individual GnRH-1 neurons (rows) in a single explant preparation. Note that some cells exhibited no detectable peaks (empty rows). The top panels (with color columns) represent the one-dimensional continuous sum of plotted cell activity (frombottom panel) in each preparation (see Materials and Methods). Asterisks indicate significant pulses of synchronized activity in the monitored population of GnRH-1 neurons. Disruption of GABA_A receptors decreased calcium peaks in individual GnRH-1 cells at both ages but only altered (lengthened) the interpulse interval in cells from 1-week-old explants.

Fig. 7.

GnRH-1 neurons express GABA_Breceptors. Representative gel documentation of PCR products from single-cell RT-PCR performed on individual cells extracted from nasal explants. (1) cDNA produced from poly(A) targeted, AL1 primer, PCR amplification; (2) products produced by PCR amplification of above cDNA using primers specific for GnRH-1 (LHRH), or (3) primers specific for GABA_B receptor.