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. 1998 Apr 1;18(7):2550-9.
doi: 10.1523/JNEUROSCI.18-07-02550.1998.

Estradiol increases dendritic spine density by reducing GABA neurotransmission in hippocampal neurons

D D Murphy  1 , N B Cole, V Greenberger, M Segal
Affiliations

Estradiol increases dendritic spine density by reducing GABA neurotransmission in hippocampal neurons

D D Murphy et al. J Neurosci. .

Abstract

We have previously shown that estradiol causes a twofold rise in dendritic spine density in cultured rat hippocampal neurons, as it does in vivo. More recently, estrogen receptors have been localized to aspiny inhibitory hippocampal interneurons, indicating that their effect on spiny pyramidal neurons may be indirect. We therefore examined the possibility that estradiol affects spine density by regulating inhibition in cultured hippocampal interneurons. Immunocytochemically, estrogen receptors were found to be co-localized with glutamate decarboxylase (GAD)-positive neurons (approximately 21% of total neurons in the culture). Exposure of cultures to estradiol for 1 d caused a marked decrease (up to 80%) in the GAD content of the interneurons, measured both by immunohistochemistry and Western blotting. Also, the number of GAD-positive neurons in the cultures decreased to 12% of the total cell population. Moreover, GABAergic miniature IPSCs were reduced in both size and frequency by estradiol, whereas miniature EPSCs increased in frequency. We then mimicked the proposed effects of estradiol by blocking GABA synthesis with mercaptopropionic acid (MA). Cultures treated with MA expressed a dose-dependent decrease in GABA immunostaining that mimicked that seen with estradiol. MA-treated cultures displayed a significant 50% increase in dendritic spine density over controls, similar to that produced by estradiol. These results indicate that estradiol decreases GABAergic inhibition in the hippocampus, which appears to effectively increase the excitatory drive on pyramidal cells, and thus may provide a mechanism for formation of new dendritic spines.

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Figures

Fig. 1.
Fig. 1.
Estrogen receptors are found in GAD-positive neurons of rat hippocampal cultures. A,B, Double staining of GAD immunoreactivity (red) and estrogen receptor (green) produce orange fluorescing cells.C, An estrogen receptor-negative pyramidal neuron surrounded by GAD-positive puncta, staining GABAergic terminals on the soma. Scale bar, 20 μm. Note GAD-positive terminals on the somata of the estrogen receptor-positive neurons, indicating that ERαs are confined to the somata and do not travel to the GAD-positive terminals.
Fig. 2.
Fig. 2.
Estradiol suppresses GAD expression in cultured hippocampal neurons. A, Control. B, Twenty-four-hour exposure to estradiol. Scale bar, 20 μm.C, Percentage of GAD-positive neurons of the total cells in all of the randomly sampled fields from control (ct) and estradiol (est)-treated cultures; estradiol cultures are different from controls using χ2 analysis;p < 0.01. D, Estradiol reduces the fluorescence intensity of GAD staining (scale in arbitrary fluorescence units); mean ± SEM. E, Western blotting of GAD-stained gels, detecting both GAD65 (bottom band) and GAD67 (top band). F, Summary of densitometry analysis of effects of estradiol on total GAD immunoreactivity shown in E. Scale is percentage of matched untreated control lanes. In D andF, asterisks denote difference from controls using t tests; p < 0.01.
Fig. 3.
Fig. 3.
GABA evokes inhibitory responses in cultured hippocampal neurons. A, Cell-attached patch recording of action potential discharges in a cultured neuron. Spontaneous activity (top 4 traces) is inhibited by exposure of the cell to 10 μm GABA applied via an adjacent pressure pipette (middle 4 traces). Spontaneous activity recovers thereafter. Bottom traces, Culture treated with estradiol for 24 hr. Patch pipette contained extracellular recording medium. B, C, Similar postsynaptic responses to GABA in control and estradiol-treated cultures.B, GABA-induced currents in control (top) and estradiol-treated (bottom) neurons. Cells were held at −60, −30, 0, and +30 mV. Patch pipette contained CsCl.C, Averaged IV curves showing similar responses to GABA of eight control (Ct) and six estradiol (Est)-treated cells for 24 hr.
Fig. 4.
Fig. 4.
Inhibitory synaptic currents evoked by glutamate application on an adjacent neuron. A brief pulse application of glutamate (top trace) evoked a series of IPSCs, responding to a series of action potentials generated in the afferent neuron. Recording was made with a patch pipette containing K-acetate. The cell was held at different potentials, and the inhibitory currents reversed near resting potential (−50 mV).
Fig. 5.
Fig. 5.
Estradiol suppresses miniature IPSCs evoked by puff application of hyperosmotic medium near the recorded cell soma.A, Control cell responding to osmotic challenge with a typical barrage of miniature IPSCs. B, Estradiol-treated cell expresses only a small series of mIPSCs. C, Frequency histogram of the sizes of the mIPSCs (in picoamperes) recorded in six control cells (404 events counted in a subset of the data for each cell). D, Frequency histogram of the total mIPSCs evoked in six estradiol-treated cells (24 hr) (210 events counted in a subset of data for each cell). E, Cumulative histogram of all the events counted in C andD, illustrating that a large proportion of medium-sized mIPSCs are absent after estradiol treatment. Cells were recorded with a patch pipette containing CsCl, and the extracellular medium also contained TTX, DNQX, and 2-APV. Cells were held at resting membrane potential (−60 mV). Ct, Control; Est, estradiol.
Fig. 6.
Fig. 6.
Estradiol effects on miniature EPSCs in cultured neurons. A, Spontaneous mEPSCs recorded from control and estradiol-treated neurons (24 hr) with a patch pipette containing CsCl. The recording medium contained TTX and 50 μm bicuculline. Cells were held at −80 mV. B, Averaged mEPSC of ∼20–30 such events in both control and estradiol-treated cells. The magnitude of synaptic current is about the same in the two cells, but its decay time constant is slightly slower in the estradiol-treated cell. C, D, Frequency distribution histograms of mEPSCs recorded in five control and six estradiol-treated cells, respectively. A sample of 20 sec was recorded from each cell. The histograms summarize a subset of 266 events recorded in the control and 506 events recorded in the treated group for an equal duration of time. The mean size of these events was −64.1 ± 2.69 pA in the control and −76.4 ± 2.95 pA in the estradiol-treated cells.E, Cumulative histograms of the events recorded in the control and estradiol-treated cells show an overall similar distribution of mEPSP sizes.
Fig. 7.
Fig. 7.
Effects of MA on GABA content and on dendritic spine formation in hippocampal cells. A–C, Control and estradiol- and MA-treated cultures, respectively, stained for GABA immunoreactivity. Scale bar, 20 μm. D–F, Control and estradiol- and MA-treated cultures, respectively, stained with DiI to reveal dendritic spines. Scale bar, 10 μm.
Fig. 8.
Fig. 8.
A, Effects of MA and estradiol (est) on GABA immunofluorescence measured in individual neurons at 24 hr. B, Effects of estradiol and MA on dendritic spine density in cultured hippocampal neurons after 48 hr. Error bars represent SE, and asterisks represent statistically significant difference from controls using Student’st tests; p < 0.01.
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