Ovarian cycle-linked plasticity of δ-GABAA receptor subunits in hippocampal interneurons affects γ oscillations in vivo

Abstract

GABAA receptors containing δ subunits (δ-GABAARs) are GABA-gated ion channels with extra- and perisynaptic localization, strong sensitivity to neurosteroids (NS), and a high degree of plasticity. In selective brain regions they are expressed on specific principal cells and interneurons (INs), and generate a tonic conductance that controls neuronal excitability and oscillations. Plasticity of δ-GABAARs in principal cells has been described during states of altered NS synthesis including acute stress, puberty, ovarian cycle, pregnancy and the postpartum period, with direct consequences on neuronal excitability and network dynamics. The defining network events implicated in cognitive function, memory formation and encoding are γ oscillations (30-120 Hz), a well-timed loop of excitation and inhibition between principal cells and PV-expressing INs (PV + INs). The δ-GABAARs of INs can modify γ oscillations, and a lower expression of δ-GABAARs on INs during pregnancy alters γ frequency recorded in vitro. The ovarian cycle is another physiological event with large fluctuations in NS levels and δ-GABAARs. Stages of the cycle are paralleled by swings in memory performance, cognitive function, and mood in both humans and rodents. Here we show δ-GABAARs changes during the mouse ovarian cycle in hippocampal cell types, with enhanced expression during diestrus in principal cells and specific INs. The plasticity of δ-GABAARs on PV-INs decreases the magnitude of γ oscillations continuously recorded in area CA1 throughout several days in vivo during diestrus and increases it during estrus. Such recurring changes in γ magnitude were not observed in non-cycling wild-type (WT) females, cycling females lacking δ-GABAARs only on PV-INs (PV-Gabrd (-/-)), and in male mice during a time course equivalent to the ovarian cycle. Our findings may explain the impaired memory and cognitive performance experienced by women with premenstrual syndrome (PMS) or premenstrual dysphoric disorder (PMDD).

Keywords: GABAA receptor; PMDD; PMS; delta subunit; gamma oscillations; ovarian cycle; parvalbumin; tonic inhibition.

FIGURE 1

Separation of behavioral states based on synchronous video and local field potential recordings. (A) Activity values plotted against the Hilbert magnitudes in the δ frequency range (1–4 Hz) for each 1 s long epoch (total: 86,400 epochs) during a full day of synchronous video-local field potential recording. Note the appearance of three clusters in the point cloud. The red rectangle delineates the point cluster corresponding to the putative REM sleep. (B) Separation of behavioral states based on combined activity and electrographic thresholds over an ∼12 min period. Bottom: local field potential recording, above: 1 s binned Hilbert magnitude of the δ-frequency range, above: activity graph, top: step function showing behavioral states based on activity and δ magnitude values (see text for details; REM, REM-sleep; NREM, NREM-sleep; Mvmt, movement; ND, not determined). For the detailed explanation of the applied calculations please refer to the Sections “Materials and Methods,” and “Electrophysiology Data Analysis.”

FIGURE 2

Fluctuations in REM sleep γ oscillation magnitudes correlate with stages of the ovarian cycle in WT mice. (A) Diagram showing average normalized 𝜃 phase coupled γ amplitudes during REM sleep on consecutive days in a WT cycling female mouse. Red and blue triangles indicate estrus and diestrus days, respectively. (B) Distribution of γ amplitudes (binned at 2 μV) recorded in all REM phases of a single day of estrus (red) and one of diestrus (blue). The same days are indicated on (A) with red (estrus) and blue (diestrus) arrows. Note the shift of the distribution toward larger values during estrus. Colored numbers indicate mean ± SEM of the corresponding histograms. (C) Top rows: 4 s long local field potential recordings with the corresponding wavelet spectra during REM sleep of estrus (top) and diestrus (bottom). (A) and (B): 𝜃 phase and γ magnitude components of local field potential segments indicated by dashed rectangles. Below: Hilbert phases of the 𝜃 (5–12 Hz) frequency range, filtered trace in the γ frequency range (63–120 Hz, black) with the corresponding Hilbert magnitudes (red). (D) FFT spectra of local field potential recordings of all REM phases during an estrus (leftmost diagram) and a diestrus day (second from left). Gray traces indicate the FFT spectra of individual REM phases, thick lines are the average FFT spectra on an estrus (red) or diestrus (blue) day. Third diagram from left shows the superimposed two average FFT spectra (estrus: red, diestrus: blue) for comparison. The area marked by dotted lines is enlarged on the diagram on the right to show the segments of the average FFT spectra where the largest deviation appears in γ activity. For the detailed explanation of the applied calculations please refer to the Sections “Materials and Methods,” and “Electrophysiology Data Analysis.”

FIGURE 3

Fluctuations in γ oscillation amplitudes recorded during REM sleep are absent in males and non-cycling WT females. (A) Left: diagram showing normalized γ amplitudes during REM sleep on consecutive days in a WT male mouse. Right: distribution of γ amplitudes (binned at 2 μV) in all REM phases recorded on two separate measurements taken 3 days apart. The 2 days are indicated on the left with red and blue arrows. Colored numbers indicate mean ± SEM of the corresponding γ amplitude histograms. (B) Summary data showing the absolute values of the differences between the normalized γ amplitudes during REM sleep in different groups of mice. In the cycling WT females the difference was calculated between days of diestrus and estrus. In WT males and non-cycling females the differences in γ amplitudes were determined at 3-day intervals that approximated the time difference between the estrus and diestrus of cycling female mice. Asterisks indicate significant difference from all other groups in a Tukey’s multiple comparison test following a one-way ANOVA.

FIGURE 4

Hippocampal δ-GABA_AR plasticity at different stages of the ovarian cycle. Representative bright field images of hippocampal DAB staining showing δ-GABA_AR expression patterns in WT cycling females at different stages of the ovarian cycle. In the hippocampus, δ-GABA_ARs are heavily expressed in the molecular layer of the dentate gyrus (DGML) and on numerous INs including neurogliaform cells and PV-INs CA1 strata oriens and radiatum (CA1 SO and SR). Expression in CA1 PC dendrites in the SR is less prominent, but noticeable. (A) δ-GABA_AR expression in DGGCs and CA1 pyramidal cells is lower in estrus compared to diestrus, and this is reflected in the staining intensity in the DGML and CA1 SO and SR. Scale bar 200 μm. (B) In CA1 and CA3 SP INs are strongly labeled with δ-GABA_AR. Optical densities of δ-GABA_AR expression were measured only in these INs (black arrowheads). Labeling of INs is weaker during estrus, which suggests lower δ-GABA_ARs expression on SP-INs (95% of which are PV-INs) during this stage of the ovarian cycle. Scale bar 50 μm. (C) Optical density measurements are in AU. Box plots represent the 25th and 75th percentile, the line in the middle is the median, and the 10th and 90th percentile are indicated by the error bars. ***p < 0.0001 difference from all other groups. (D) Optical density measurements of CA1 and CA3 SP INs in slices of cycling WT female mice (C) in diestrus (D) or estrus (E), male mice () and non-cycling WT female mice (NC). δ-GABA_ARs expression on SP-INs during estrus is lower than any other group in both CA1 and CA3 (***p < 0.0001); in the CA1 the two NC groups are both higher than any other group in CA1 (^∗p < 0.0001). Significance levels were established by one-way ANOVA followed by Tukey’s multiple comparisons test. All sections for the separate measurements in (C) and (D) were processed together to allow for densitometric comparisons.

FIGURE 5

Female PV-Gabrd^-/- mice cycle regularly but lack ovarian cycle-dependent modulation of REM phase γ oscillation amplitudes. (A) Ovarian cycle stage was determined by vaginal impedance measurements and cytological analysis of vaginal smears. PV-Gabrd^-/- mice had a similar cytological panel to WT mice at different stages. Diestrus was determined by the presence in the smear of abundant mucus, small parabasal cells (red arrowheads) and large intermediate cells (yellow arrowheads). Both cell types are absent in estrus, when smears are typically characterized by large polygonal superficial cells, mostly fully cornified. Scale bar 5 μm. (B) Left: diagram showing normalized γ amplitudes during REM sleep on consecutive days in a regularly cycling PV-Gabrd^-/- female. Red and blue triangles indicate estrus and diestrus, respectively. Right: distribution of γ amplitudes (binned at 2 μV) recorded during all REM sleep episodes for an entire day of estrus (red) and one of diestrus (blue). The 2 days are marked on the left with red (estrus) and blue (diestrus) arrows. Colored numbers indicate mean ± SEM of the corresponding histograms. Note the narrow variance of the γ amplitude distributions during both phases of the ovarian cycle.