Estradiol increases spine density and NMDA-dependent Ca²⁺ transients in spines of CA1 pyramidal neurons from hippocampal slices

To investigate the physiological consequences of the increase in spine density induced by estradiol in pyramidal neurons of the hippocampus, we performed simultaneous whole cell recordings and Ca²⁺ imaging in CA1 neuron spines and dendrites in hippocampal slices. Four- to eight-days in vitro slice cultures were exposed to 17β-estradiol (EST) for an additional 4- to 8-day period, and spine density was assessed by confocal microscopy of DiI- labeled CA1 pyramidal neurons. Spine density was doubled in both apical and basal dendrites of the CA1 region in EST-treated slices; consistently, a reduction in cell input resistance was observed in EST-treated CA1 neurons. Double immunofluorescence staining of presynaptic (synaptophysin) and postsynaptic (α-subunit of CaMKII) proteins showed an increase in synaptic density after EST treatment. The slopes of the input/output curves of both α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and N-methyl-D- aspartate (NMDA) postsynaptic currents were steeper in EST-treated CA1 neurons, consistent with the observed increase in synapse density. To characterize NMDA-dependent synaptic currents and dendritic Ca²⁺ transients during Schaffer collaterals stimulation, neurons were maintained at +40 mV in the presence of nimodipine, picrotoxin, and 6-cyano-7-nitroquinoxaline-2,3- dione (CNQX). No differences in resting spine or dendritic Ca²⁺ levels were observed between control and EST-treated CA1 neurons. Intracellular Ca²⁺ transients during afferent stimulation exhibited a faster slope and reached higher levels in spines than in adjacent dendrites. Peak Ca²⁺ levels were larger in both spines and dendrites of EST-treated CA1 neurons. Ca²⁺ gradients between spine heads and dendrites during afferent stimulation were also larger in EST-treated neurons. Both spine and dendritic Ca²⁺ transients during afferent stimulation were reversibly blocked by D,L-2- amino-5-phosphonovaleric acid (D,L-APV). The increase in spine density and the enhanced NMDA-dependent Ca²⁺ signals in spines and dendrites induced by EST may underlie a threshold reduction for induction of NMDA-dependent synaptic plasticity in the hippocampus.