Heterogeneity in the basic membrane properties of postnatal gonadotropin-releasing hormone neurons in the mouse

Abstract

The electrophysiological characteristics of unmodified, postnatal gonadotropin-releasing hormone (GnRH) neurons in the female mouse were studied using whole-cell recordings and single-cell RT-PCR methodology. The GnRH neurons of adult animals fired action potentials and exhibited distinguishable voltage-current relationships in response to hyperpolarizing and depolarizing current injections. On the basis of their patterns of inward rectification, rebound depolarization, and ability to fire repetitively, GnRH neurons in intact adult females were categorized into four cell types (type I, 48%; type II, 36%; type III, 11%; type IV, 5%). The GnRH neurons of juvenile animals (15-22 d) exhibited passive membrane properties similar to those of adult GnRH neurons, although only type I (61%) and type II (7%) cells were encountered, in addition to a group of "silent-type" GnRH neurons (32%) that were unable to fire action potentials. A massive, action potential-independent tonic GABA input, signaling through the GABA(A) receptor, was present at all ages. Afterdepolarization and afterhyperpolarization potentials (AHPs) were observed after single action potentials in subpopulations of each GnRH neuron type. Tetrodotoxin (TTX)-independent calcium spikes, as well as AHPs, were encountered more frequently in juvenile GnRH neurons compared with adults. These observations demonstrate the existence of multiple layers of functional heterogeneity in the firing properties of GnRH neurons. Together with pharmacological experiments, these findings suggest that potassium and calcium channels are expressed in a differential manner within the GnRH phenotype. This heterogeneity occurs in a development-specific manner and may underlie the functional maturation and diversity of this unique neuronal phenotype.

Fig. 1.

A, High-power photomicrograph of a patched bipolar-type neuron (asterisk) located in the rostral preoptic area subsequently proven to express GnRH transcripts.B, Gel showing the presence of 213 bp GnRH amplicons in cells 1, 2, 4, and 5. DNA 1 kb ladder is to theright.

Fig. 2.

Spontaneous synaptic activity recorded in GnRH neurons from a 55-d-old female mouse. Top trace shows a continuous trace of an experiment in which TTX (0.5 μm) and bicuculline (10 μm) were applied to a GnRH neuron at its resting membrane potential of −70 mV. The bottom traces are expanded 1 sec traces recorded in control (a), the presence of TTX (b), the presence of both bicuculline and TTX (c), and after washout (d).

Fig. 3.

Intrinsic membrane properties of four types (I–IV) of GnRH neurons recorded in adult female mice.A–D, Responses to 20 and 200 msec depolarizing and hyperpolarizing current pulses. In all four cell types, threshold stimulation elicited a single action potential, which clearly shows depolarizing afterpotential (ADP) of various duration and amplitude. In each cell type, 200 msec duration hyperpolarizing current injection revealed the presence of inward rectifiers with distinct kinetics. The four cell types are characterized on the basis of their inward rectification as well as the absence of rebound depolarization in type II cells and the inability to fire repetitively after depolarization in type III cells. Resting membrane potentials are given for each cell.

Fig. 4.

Intrinsic membrane properties of GnRH neurons recorded in juvenile female mice. A, Type I neuron in which action potentials could be evoked with 20 and 200 msec depolarizing and hyperpolarizing current pulses. Voltage–current relationship plotted from the end of 200 msec duration pulses. Note the presence of afterhyperpolarization with the 20 msec depolarizing pulse.B, Silent-type neuron in which no action potential could be evoked with either 20 or 200 msec depolarizing and hyperpolarizing current pulses. Voltage–current relationship plotted from the end of 200 msec pulse. The resting membrane potential was −67 mV in cellA and −72 mV in cell B.

Fig. 5.

The effect of tetrodotoxin (TTX) on cell type I in adult and juvenile GnRH neurons. Families of voltage were evoked in adult GnRH (Aa) and in juvenile GnRH neurons (Ba) with 200 msec current pulses in control and in the presence of TTX. The corresponding voltage–current relationship obtained from the adult GnRH neuron (Ab) shows marked rectification with depolarizing pulses. Note the presence of an “A” notch (arrow) in the presence of TTX. Bb, Expanded traces in response to 0.06 nA depolarizing current pulse, showing that in the presence of TTX, calcium spikes are revealed. Theinsets illustrate the differences in the shape and duration of sodium (no TTX) and calcium (in TTX) spikes. The resting membrane potential is −74 mV in cell A and −74 mV in cell B.

Fig. 6.

The effect of tetraethylammonium (TEA) and 4-aminopyridine (4-AP) on the spike discharge of adult GnRH neurons. A, Families of voltage were evoked in cell type I, with 200 msec hyperpolarizing and depolarizing current pulses in the presence and absence of 1 mm TEA. TEA decreased cell excitability in GnRH neurons, and as shown by the inset, it produced this effect by broadening the action potential. B, Responses to 200 msec depolarizing and hyperpolarizing current pulses in cell type IV in the presence of 100 μm 4-AP. 4-AP produced an increase in cell excitability by altering the repolarization phase of the action potential as shown in the inset. The resting membrane potential is −70 mV in cell A and −74 mV in cellB.

Fig. 7.

The effect of barium on GnRH neurons. Responses to 200 msec depolarizing and hyperpolarizing current pulses evoked in a silent-type juvenile GnRH neuron in control, on addition of 100 μm barium and after washout. The corresponding voltage–current relationships at the beginning (30 msec, ○) and end (190 msec, ●) are plotted, revealing the presence of rectification with depolarizing and hyperpolarizing pulses. Note the blockade of both inward and outward rectification in Ba²⁺, which was readily reversible. Resting membrane potential was −65 mV but brought to −70 mV with DC.

Fig. 8.

The effect of cesium on type III (A) and type IV (B) GnRH neurons. Families of voltage are evoked with 200 msec depolarizing and hyperpolarizing current pulses in control and in the presence of 100 or 300 μm Cs⁺. Note the blockade of anomolous rectification (A) andI_Q/H (B) by Cs⁺, without any effect on cell excitability. Voltage–current relationship plotted from the end of the 200 msec pulse in control (○) and in the presence of cesium (●). Resting membrane potential was −72 mV in A and −75 mV inB.