Electrical and synaptic properties of embryonic luteinizing hormone-releasing hormone neurons in explant cultures.

Abstract

Voltage-and ligand-activated channels in embryonic neurons containing luteinizing hormone-releasing hormone (LHRH) were studied by patch-pipette, whole-cell current and voltage clamp techniques. LHRH In mammals, luteinizing hormone (LH) pulses are closely correlated with increased activities in as yet unidentified mediobasal hypothalamic neurons (1-3) which are believed to regulate the secretory activities of LH-releasing hormone (LHRH)-containing neurons. Due to the small number of LHRH neurons in brain, their small diameter, and their dispersed anatomical distribution, very few electrical recordings from identified LHRH neurons either in vivo or in vitro are available (4, 5). Consequently, the underlying electrophysiological and synaptic properties of LHRH neurons and information about their basic physiological properties have been largely deduced from extensive studies on the immortalized hypothalamic (LHRH-cell) neuronal cell line GT1 (6). Cells of the GT1-3 and -7 sublines possess a rich array of ion channels (7-11) and, under some circumstances, exhibit pulsatile LHRH secretion (9,(11)(12)(13)(14). However, the extent to which these electrical, receptor, and secretory properties of GT1 cells are found in actual LHRH neurons is unknown.Our aim was to study the electrophysiological and synaptic properties of primary LHRH neurons. Ontogenetic studies have shown that LHRH neurons originate from progenitor cells in the olfactory placode and thereafter migrate from the olfactory pit (OP) into the forebrain during prenatal development (15,16). LHRH neurons can be cultured from embryonic olfactory placodal regions (17, 18), and cells maintained in these explants secrete LHRH in a pulsatile mannerThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 3918(19). We report here successful intracellular recordings from 62 identified embryonic LHRH neurons and describe their highly differentiated voltage-and ligand-gated ion-channel properties.MATERIALS AND METHODS Preparations. OP regions ("0.5 mm3) were aseptically dissected from day-11.5 mouse embryos (17), plated on glass coverslips (24 mm x 12 mm, Gold Seal, no. 1 thickness), and placed in 35-mm Petri dishes (Falcon). Tissues were fed twice a week with serum-free Eagle's basal medium (20) and maintained in humidified air plus 5% CO2. Cultures typically received a single dose of 5-fluoro-2'-deoxyuridine (80 ,uM) on day 3 for 3 days. This treatment reduced the proliferation of dividing cells and vastly improved the visualization of LHRHlike (bipolar) neurons under phase-contrast microscopy (see Fig. 1). Tissues were maintained for 6-14 days in vitro, during which time LHRH cells migrated into the thin peripheral tissue areas (17), thereby facilitating electrophysiological recordings.Electrophysiological Recordings. The cultures on coverslips were transferred to a recording chamber on a Nikon M inverted mi...

Physiologic Regulation of a Tetrodotoxin-Sensitive Sodium Influx That Mediates a Slow Afterdepolarization Potential in Gonadotropin-Releasing Hormone Neurons: Possible Implications for the Central Regulation of Fertility

“…1 B). The small amount of current required to initiate action potential firing is consistent with the high input resistance of these cells (Kusano et al, 1995;Spergel et al, 1999;Suter et al, 2000b;DeFazio and Moenter, 2002;Kuehl-Kovarik et al, 2002). There was little evidence of spike frequency adaptation over the 500 ms injection, although a slight decrement in spike amplitude was routinely observed.…”

Direct Action of Estradiol on Gonadotropin-Releasing Hormone-1 Neuronal Activity via a Transcription-Dependent Mechanism

“…Therefore, taking advantage of the extra-CNS origin of these cells, several groups have established an in vitro model of postmitotic, primary GnRH-1 cells in nasal explants. Individual GnRH-1 neurons in nasal explants exhibit electrical and synaptic properties similar to in vivo GnRH-1 neurons, including responses to GABAergic and glutamatergic stimulation (Kusano et al, 1995). In addition to depolarized-induced GnRH-1 release (Terasawa et al, 1999a;Moore and Wray, 2000), as in vivo, GnRH-1 neurons in nasal explants release GnRH-1 in a pulsatile manner (Terasawa et al, 1999a;Funabashi et al, 2000) with the interpulse interval being species dependent.…”

Heterogeneity in the Basic Membrane Properties of Postnatal Gonadotropin-Releasing Hormone Neurons in the Mouse

“…Previous electrophysiological studies have demonstrated the presence of I K , I A , and I IR , as well as low-and high-thresholdactivated calcium conductances, in immortalized GT1 cells (Bosma, 1993;Hales et al, 1994;Van Goor et al, 1999a,b) and cultured embryonic GnRH neurons of the mouse (Kusano et al, 1995). Although detailed information on the basic membrane properties of green fluorescent protein (GFP)-expressing GnRH neurons is not yet available, it is interesting to note that outward potassium currents indicative of I A and I K conductances were reported by Spergel and colleagues (1999).…”