A primary cell culture system of luteinizing hormone releasing hormone neurons derived from embryonic olfactory placode in the rhesus monkey

Abstract

The purpose of this study is to establish a primary LHRH cell culture system using embryonic olfactory placode and to examine whether LHRH cells derived from olfactory placode and the migratory pathway of LHRH neurons mature in vitro. Six monkey fetuses at the ages of E34-E36 were delivered surgically and the area including the olfactory placode (PL) and the areas that encompass the migratory pathway (MP) were dissected out. The tissues were cut into small pieces and plated on collagen- or poly-L-lysine-coated glass coverslips in medium M199. Cultures were maintained for up to 33 days and immunostained for LHRH, GnRH-associated peptide, neurofilament protein, neuron-specific enolase, and glial fibrillary acidic protein. LHRH positive cells were also positive for neurofilament proteins neuron-specific enolase, and GnRH-associated peptides, but negative for glial fibrillary acidic protein. In the first week of culture, LHRH cells remained within the explants of PL, were rounded (average dimensions: 13.0 x 11.3 microns) and stained lightly. By the second week a number of LHRH cells (15.7 x 13.6 microns) with neurites started to migrate out from PL explants, whereas some still remained in the PL. By the third week a large number of LHRH cells (19.3 x 9.4 microns) had migrated out from the PL. They were fusiform in shape with clear nuclei and extended long varicose neurites up to 500 microns in length. A few "pioneer" LHRH cells appeared to lead the migration of 100-400 LHRH cells forming 1-3 major migratory paths. In contrast, LHRH cells from MP explants migrated out sooner than those from PL explants. LHRH cells from the ventral part of the MP, which is close to the PL, migrated out by 1-2 weeks and formed several migratory paths, whereas LHRH cells from the dorsal part of the MP, which is farther from the PL, were scattered widely around explants and their neurites were extended tortuously. Cultured LHRH cells released LHRH into the media and responded to challenge with high K+. The results indicate that 1) primary LHRH neurons can be obtained from the embryonic PL and their migratory pathway, 2) these neurons migrate and mature in culture and 3) they are accessible for cellular and molecular studies.

In VivoRecordings of GnRH Neuron Firing Reveal Heterogeneity and Dependence upon GABAAReceptor Signaling

“…This suggests that the firing patterns and dynamics recorded here from GnRH neurons in vivo are not impacted substantially by pentobarbital anesthesia. Of note, the pentobarbital suppression of pulsatile LH secretion in vivo in rats results from the ensuing hypothermia rather than pentobarbital anesthesia itself (Strutton and Coen, 1996).…”

Involvement of headless myosin X in the motility of immortalized gonadotropin‐releasing hormone neuronal cells

“… Uncited references Duittoz et al, 1997; Murase and Horwitz, 2002; Terasawa et al, 1993; Tokuo and Ikebe, 2004. …”

Neuroestrogen, rapid action of estradiol, and GnRH neurons

“…Because transgenic monkeys with GFP-labeled GnRH neurons are not yet available, this approach is quite useful for studying the cellular physiology of GnRH neurons in primates. Earlier, we have demonstrated that 1) cultured GnRH neurons from monkeys contain almost no non-GnRH neurons or glia, as they are not present in the nasal placode at this specific developmental stage (146), 2) cultured GnRH neurons undergo maturational changes in vitro (79,146), and 3) placode derived GnRH neurons are functional, as transplantation of fetal placode into the infundibular recess of the third ventricle of adult female monkeys, in which the GnRH final common pathway has been lesioned, restores cyclic ovulation (125). …”

“…Because our GnRH cultures contain non-neuronal cells (146) and they are involved in the propagation of the [Ca 2+ ] i wave of the GnRH neuronal network (121), it is possible that E 2 action to GnRH neurons are also mediated through non-neuronal cells. In fact, E 2 rapidly induces a [Ca 2+ ] i release from astrocytes, stimulates progesterone synthesis within 5 min in astrocytes of rat hypothalamus which are dependent upon both nonclassical and classical receptors (16,78), and modifies tanycyte morphology (70,113).…”