Estradiol modulation of phenylephrine-induced excitatory responses in ventromedial hypothalamic neurons of female rats

Abstract

Estrogens act within the ventromedial nucleus of the hypothalamus (VMN) to facilitate lordosis behavior. Estradiol treatment in vivo induces alpha(1b)-adrenoreceptor mRNA and increases the density of alpha(1B)-adrenoreceptor binding in the hypothalamus. Activation of hypothalamic alpha(1)-adrenoceptors also facilitates estrogen-dependent lordosis. To investigate the cellular mechanisms of adrenergic effects on VMN neurons, whole-cell patch-clamp recordings were carried out on hypothalamic slices from control and estradiol-treated female rats. In control slices, bath application of the alpha(1)-agonist phenylephrine (PHE; 10 microM) depolarized 10 of 25 neurons (40%), hyperpolarized three neurons (12%), and had no effect on 12 neurons (48%). The depolarization was associated with decreased membrane conductance, and this current had a reversal potential close to the K(+) equilibrium potential. The alpha(1b)-receptor antagonist chloroethylclonidine (10 microM) blocked the depolarization produced by PHE in all cells. From estradiol-treated rats, significantly more neurons in slices depolarized (71%) and fewer neurons showed no response (17%) to PHE. PHE-induced depolarizations were significantly attenuated with 4-aminopyridine (5 mM) but unaffected by tetraethylammonium chloride (20 mM) or blockers of Na(+) and Ca(2+) channels. These data indicate that alpha(1)-adrenoceptors depolarize VMN neurons by reducing membrane conductance for K(+). Estradiol amplifies alpha(1b)-adrenergic signaling by increasing the proportion of VMN neurons that respond to stimulation of alpha(1b)-adrenergic receptors, which is expected in turn to promote lordosis.

Fig. 1.

PHE-induced membrane depolarization in VMN neurons in the current-clamp mode. (A) PHE caused a reversible depolarization of the membrane potential in a VMN neuron. (B) PHE depolarized and induced firing in another VMN neuron. Action potentials were truncated. (C) PHE caused a reversible depolarization in a VMN neuron. After washout of PHE, the presence of the α1b-adrenoreceptor antagonist chloroethylclonidine (10 μM) blocked PHE-induced depolarization in the same neuron.

Fig. 2.

Current–voltage relationship before and after PHE application. (A) Current–voltage (I–V) relationship during baseline (•) and PHE (▵) application. The I–V relationship shows a decrease in the slope in the presence of PHE, indicating a decrease in membrane conductance. (B) Net PHE currents obtained by subtractions of 2 − 1 for the corresponding I–V curves shown in A. The reversal potential was estimated to be −96 mV, close to that for potassium.

Fig. 3.

Estradiol treatment increases the proportion of neurons depolarizing in response to PHE. Histograms show the proportion of VMN neurons responding to 10 μM PHE was higher in estradiol-treated (Right) than control (Left) neurons. The numbers of neurons tested are noted. *, P < 0.03.

Fig. 4.

PHE-induced membrane depolarization is significantly reduced in the presence of 4-AP. (A) Sample traces of current clamp recordings from six VMN neurons showing PHE response to ion channel blockers and ion substitution. PHE response remained in the presence of TTX (0.5 μM), BAPTA (10 or 20 mM) in the internal pipette solution, cadmium (100 μM), calcium-free ACSF, and TEA (20 mM). However, 4-AP (5 mM) significantly blocked the excitatory action of PHE. (B) PHE depolarized the membrane potential of a VMN neuron. After washout of PHE, the neuron was reexposed to PHE in the presence of 4-AP. 4-AP completely blocked PHE-induced depolarization in the same neuron. (C) Membrane depolarization in response to PHE in regular ACSF, and after washout, in ACSF with 4-AP in six neurons. 4-AP completely blocked PHE-induced depolarization in four neurons and reduced depolarization in two neurons.