Episodic bursting activity and response to excitatory amino acids in acutely dissociated gonadotropin-releasing hormone neurons genetically targeted with green fluorescent protein

Abstract

The gonadotropin-releasing hormone (GnRH) system, considered to be the final common pathway for the control of reproduction, has been difficult to study because of a lack of distinguishing characteristics and the scattered distribution of neurons. The development of a transgenic mouse in which the GnRH promoter drives expression of enhanced green fluorescent protein (EGFP) has provided the opportunity to perform electrophysiological studies of GnRH neurons. In this study, neurons were dissociated from brain slices prepared from prepubertal female GnRH-EGFP mice. Both current- and voltage-clamp recordings were obtained from acutely dissociated GnRH neurons identified on the basis of EGFP expression. Most isolated GnRH-EGFP neurons fired spontaneous action potentials (recorded in cell-attached or whole-cell mode) that typically consisted of brief bursts (2-20 Hz) separated by 1-10 sec. At more negative resting potentials, GnRH-EGFP neurons exhibited oscillations in membrane potential, which could lead to bursting episodes lasting from seconds to minutes. These bursting episodes were often separated by minutes of inactivity. Rapid application of glutamate or NMDA increased firing activity in all neurons and usually generated small inward currents (<15 pA), although larger currents were evoked in the remaining neurons. Both AMPA and NMDA receptors mediated the glutamate-evoked inward currents. These results suggest that isolated GnRH-EGFP neurons from juvenile mice can generate episodes of repetitive burst discharges that may underlie the pulsatile secretion of GnRH, and glutamatergic inputs may contribute to the activation of endogenous bursts.

Fig. 1.

Dissociation of GnRH-EGFP neurons from brain slices. Neurons were isolated from 400-μm-thick hypothalamic slices that included the diagonal band of Broca (DBB) and surrounding septum (A, Rostral) or the medial preoptic area (MPA) and surrounding hypothalamus (B, Caudal). Two to three slices were taken per animal. Dark gray indicates the area with the highest concentration of GnRH neurons, although cells are also scattered throughout surrounding areas (light gray). Dark lines represent the cuts made to isolate the region before enzymatic treatment. Atlas figures were modified from Franklin and Paxinos (1997) with permission.ac, Anterior commissure; BST, bed nucleus of the stria terminalis; HDB, horizontal limb of the diagonal band of Broca; ic, internal capsule;LPO, lateral preoptic area; LS, lateral septum; LV, lateral ventricle; MS, medial septum; VP, ventral pallidum.

Fig. 2.

Identification of isolated GnRH-EGFP neurons.A₁, Phase-contrast image of an isolated, live GnRH-EGFP neuron. Note the round, phase-bright appearance and cellular debris. A₂, Fluorescence view of the same GnRH-EGFP neuron. Scale bar, 20 μm. Images were captured at 40× with a confocal microscope (Zeiss) 18 hr after dissociation. B₁, Differential interference contrast image of neurons after acute dissociation. The same field was visualized with a GFP filter (Chroma Technology Corp., Brattleboro, VT) (B₂) and a rhodamine filter (B₃). Note that only one neuron shows EGFP fluorescence, and that same neuron is the only one immunoreactive for GnRH (B₄). Images were captured at 40× with a Zeiss Axioplan 2 imaging system. Scale bar, 50 μm.

Fig. 3.

Spontaneous bursting activity. Representative patterns of repetitive bursts in cell-attached mode (A) and in whole-cell mode (B). Both traces demonstrate repetitive bursts. C, A high-gain, filtered trace of a region in B (indicated by the bar) demonstrating the oscillation of membrane potential that accompanied each burst. The baseline indicates −60 mV in B andC. The action potentials in C are cropped, and filtering resulted in the clipping of an action potential (arrow). A and B represent 3 min of activity; C represents 18 sec. The neuron inA was obtained from a P21 animal. B andC were from a P17 animal.

Fig. 4.

Episodic activity. A,B, Ten minute traces from two neurons demonstrating that spontaneous bursts can occur in episodes of activity that are separated by minutes of silence. The neuron in B was more active (see histogram in Fig. 5B). A represents the region indicated by the bar in the histogram in Figure 5A, and B represents the region in Figure 5B. Both neurons were hyperpolarized to −75 mV by current injection. Note the long periods of inactivity, the multiple bursts, and the oscillations in membrane potential in bothtraces. The data shown in A was obtained from a P17 mouse (same neuron as in Fig.3B,C), whereas the data shown in B was obtained from a P21 mouse.

Fig. 5.

Variability in episodic activity. Frequency histograms illustrating spontaneous episodic activity in six GnRH-EGFP neurons (25 min of recording is shown in A–E and 5 min in F). The number of events in each 6 sec interval is plotted versus time. Note the variability in activity level among neurons. Also note multiple episodes of bursting separated by periods of inactivity extending up to 20 min. The duration of episodic activity in A–E ranged from 28 to 70 min, and length of record in F was 11 min. Raw data from the neurons shown in A and B (hatched bars) were also presented in Figure 4. All recordings were from P17–P23 mice: A, P17; B, P21; C, P21 (same animal as B); D, P22;E, P23; F, P21.

Fig. 6.

Membrane properties of GnRH-EGFP neurons.A, Typical responses to hyperpolarizing and depolarizing 200 msec current pulses of −10, −20, −30, −40, −50, and −60 pA and +10, +20, +30, and +40 pA. This neuron had a resting membrane potential of −58 mV. Responses to hyperpolarization did not rectify and, in this example, demonstrated a V_{20 msec}/V_{190 msec} ratio of 0.49. Depolarization resulted in the firing of a train of action potentials.B, Waveform of a typical action potential, taken from the same neuron as shown above, during a 20 pA depolarizing current pulse of 200 msec duration.

Fig. 7.

Glutamate- and NMDA-evoked activity. Representative patterns of activity recorded in current-clamp mode during application of 10 mm glutamate (A₁,B₁) or 300 μm NMDA (A₂,B₂).A₁ andA₂ are depolarizations observed in a GnRH-EGFP neuron with a small-amplitude current response (5 pA) to 10 mm glutamate (see Fig. 8A).B₁ andB₂ are depolarizations from a neuron with a larger (80 pA) current response. Application of agonist increased firing and depolarized the membrane in all cases. Neurons were isolated from the brain of a P23 mouse.

Fig. 8.

Excitatory amino acid-evoked currents.A, Representative inward currents evoked by the 500 msec application of 10 mm glutamate or 300 μmNMDA. Currents were recorded from a GnRH-EGFP neuron lifted off the bottom of the culture dish. This neuron was isolated from a P23 mouse. Each trace represents a single application of agonist.B, Inward current evoked by the 500 msec application of 10 mm glutamate (GLU), superimposed on the response to 10 mm glutamate and 100 μmcyclothiazide (CTZ). Note the large potentiation and absence of desensitization of the glutamate response by cyclothiazide. Currents were recorded from a GnRH-EGFP neuron that was attached to the bottom of the dish. The neuron was isolated from the brain of a P17 mouse. Traces are the average of 5–10 records.C, Frequency histogram illustrates the response amplitude to glutamate in 34 cells. Note the large number of neurons with a small (<15 pA) response.

Fig. 9.

Hypothetical relationship between action potentials of isolated GnRH neurons and multiple-unit activity of the GnRH population responsible for pulsatile release of GnRH. It is hypothesized that GnRH pulsatility arises from overlapping episodes of bursts of action potentials in many GnRH neurons. A, Schematic diagram depicting three possible models for how GnRH neurons could fire as a network: independently (Terasawa, 2001), coupled (Witkin et al., 1995; Hosny and Jennes, 1998; Hu et al., 1999), or triggered by one or a few divergent neurons with pulse-generating properties (van den Pol and Trombley, 1993; Boudaba et al., 1997). In all three cases, the electrical activity will cause hormone release at the median eminence. GnRH neurons are depicted as filled circles; the pulse-generating cell is depicted as afilled square; arrows indicate pulses of GnRH release. B, The binned activities of five independent neurons (taken from Fig. 5) were summed into a single profile. The most active neuron (Fig. 5B) dominates the summed activity, and the other four cells also shape its profile. “Pulses” of activity emerge from this profile. C, The data in B were smoothed by calculating the moving average of 20 data points around each data point. We speculate that the smoothing function could represent an averaging of the activity of 800–1000 GnRH neurons in the hypothalamus and would yield pulses of electrical activity that mimic the resultant pulsatile release of hormone. In this scheme, the dotted line represents a baseline level of hormone that would be constitutively released, and the three major waves of activity represent pulsatile hormone release. The arrows indicate where, in the COUPLEDmodel (A₂), there would be synchronized activity, or, in the TRIGGERED model (A₃), the release of glutamate onto many GnRH neurons would initiate a pulse of activity and hormone release. The averaged data in C, however, actually represent the INDEPENDENT activity of five neurons, as depicted in A₁.