Dual Somatic Recordings from Gonadotropin-Releasing Hormone (GnRH) Neurons Identified by Green Fluorescent Protein (GFP) in Hypothalamic Slices

Abstract

Gonadotropin-Releasing Hormone (GnRH) is a small neuropeptide that regulates pituitary release of luteinizing hormone (LH) and folliclestimulating hormone (FSH). These gonadotropins are essential for the regulation of reproductive function. The GnRH-containing neurons are distributed diffusely throughout the hypothalamus and project to the median eminence where they release GnRH from their axon terminals into the hypophysiotropic portal system (1). In the portal capillaries, GnRH travels to the anterior pituitary gland to stimulate release of gonadotropins into systemic circulation. GnRH release is not continuous but rather occurs in episodic pulses. It is well established that the intermittent manner of GnRH release is essential for reproduction (2, 3).Coordination of activity of multiple GnRH neurons probably underlies GnRH pulses. Total peptide content in GnRH neurons is approximately 1.0 pg/cell (4), of which 30% likely comprises the releasable pool. Levels of GnRH during a pulse (5, 6), suggest multiple GnRH neurons are probably involved in neurosecretion. Likewise, single unit activity extracted from hypothalamic multi-unit recordings during LH release indicates changes in activity of multiple neurons (7). The electrodes with recorded activity during LH pulses are associated with either GnRH somata or fibers (8). Therefore, at least some of this activity arises from GnRH neurons.The mechanisms that result in synchronized firing in hypothalamic GnRH neurons are unknown. Elucidating the mechanisms that coordinate firing in GnRH neurons is a complex problem. First, the GnRH neurons are relatively few in number. In rodents, there are 800-2500 GnRH neurons. It is not clear that all GnRH neurons are involved in episodic GnRH release. Moreover, GnRH neurons are diffusely distributed (1). This has complicated our understanding of coordination of firing and has made many technical approaches intractable. We have optimized loose cell-attached recordings in current-clamp mode for the direct detection of action potentials and developed a recording approach that allows for simultaneous recordings from pairs of GnRH neurons. Video LinkThe video component of this article can be found at http://www.jove.com/video/1678/ Protocol 1. Hypothalamic brain slices are prepared, incubated and transferred to the recording chamber as previously detailed (9) in animals whose GnRH neurons express green fluorescent protein (GFP) under the control of the GnRH peptide promoter (10). After removal of the brain from the animal, the brain is blocked using a razor blade to eliminate regions that are not of interest. In order to determine the regions to remove, the brain is placed on its dorsal surface so that the hypothalamus can be visualized. For both the coronal and sagittal slice orientations, the cerebellum is removed. For coronal slice preparation, the rostral portion of the cortex is removed. In this slice orientation, the lateral regions of the cortex provide support for slicing the brain and provide sufficient area of brain ...

Simulated GABA Synaptic Input and L-Type Calcium Channels Form Functional Microdomains in Hypothalamic Gonadotropin-Releasing Hormone Neurons

“…We have previously validated our cell-attached recording approach for the detection of action potentials with low resistance seals (~30–50 MΩ; Hemond and Suter 2010). We used Axon Instrument’s 2B Axoclamp (Axon Instruments; Union City, CA) in current clamp mode as the primary amplifier in series with a second amplifier (AM Systems 3000) to increase signal detection and to allow for detection of action potentials.…”

Hormone secretion in transgenic rats and electrophysiological activity in their gonadotropin releasing-hormone neurons

“…We used the cell-attached mode with low-resistance seals (ϳ30 -50 M⍀). We have previously validated our long-term recording approach using Axon Instrument's 2B Axoclamp (Axon Instruments, Union City, CA) in current clamp mode as the primary amplifier used in series with a second amplifier (AM Systems 3000) to increase signal detection and allow for detection of action potentials (12). Data were acquired at 10 kHz using p-Clamp software (version 10.0; Molecular Devices, Union City, CA).…”