Hippocampal excitability increases during the estrous cycle in the rat: a potential role for brain-derived neurotrophic factor

Abstract

To test the hypothesis that induction of BDNF may contribute to changes in hippocampal excitability occurring during the female reproductive cycle, we examined the distribution of BDNF immunoreactivity and changes in CA1 and CA3 electrophysiology across the estrous cycle in rats. Hippocampal BDNF immunoreactivity increased on the day of proestrus as well as on the following morning (estrus), relative to metestrus or ovariectomized animals. Changes in immunoreactivity were clearest in mossy fiber axons of dentate gyrus granule cells, which contain the highest concentration of BDNF. Increased immunoreactivity was also apparent in the neuropil-containing dendrites of CA1 and CA3 neurons. Electrophysiological recordings in hippocampal slices showed robust cycle-dependent differences. Evoked responses of CA1 neurons to Schaffer collateral stimulation changed over the cycle, with larger maximum responses at both proestrus and estrus relative to metestrus. In area CA3, repetitive hilar stimuli frequently evoked multiple population spikes at proestrus and estrus but only rarely at other cycle stages, and never in slices of ovariectomized rats. Hyperexcitability in area CA3 at proestrus was blocked by exposure to the high-affinity neurotrophin receptor antagonist K252a, or an antagonist of the alpha7 nicotinic cholinergic receptor, whereas it was induced at metestrus by the addition of BDNF to hippocampal slices. These studies suggest that hippocampal BDNF levels change across the estrous cycle, accompanied by neurophysiological responses that resemble the effects of BDNF treatment. An estrogen-induced interaction of BDNF and alpha7 nicotinic receptors on mossy fibers seems responsible for estrous cycle changes in area CA3. Periovulatory changes in hippocampal function may, thus, involve estrogen-induced increases in BDNF expression.

Figure 3.

Changes in evoked responses of area CA1 pyramidal cells during the estrous cycle. A, Diagram of the Schaffer collaterals stimulating site (SCH) and recording site in the CA1b pyramidal cell layer (x). B, Representative evoked responses to half-maximal stimulus at metestrus (top) and estrus (bottom). For this figure and others, stimulus artifacts are marked by dots and are truncated. C, Population spike amplitude is plotted as a function of stimulus intensity. The stimulus was a rectangular 100 μA pulse, and stimulus intensity was adjusted by changing stimulus duration in 10 μsec intervals. Fourteen animals were studied (proestrus, n = 5; estrus, n = 4; metestrus, n = 5). Two to four slices per animal were used, and results for all slices of a given animal were averaged. Results were then averaged across animals. Data represent means ± SEM of the results for each stimulus duration, at each stage of the cycle. The lines drawn through the points represent the best-fit curves generated by computer-assisted least-squares analysis (see Materials and Methods).

Figure 4.

Electrophysiological changes in area CA3 during the estrous cycle. A, Diagram of recording (filled circles) and stimulation sites (x) for data shown here and in Figures 5, 6, 7, 8, 9, 10, 11. PCL, Pyramidal cell layer; GCL, granule cell layer; SCH, Schaffer collaterals; FIM, fimbria; SR, stratum radiatum; SL, stratum lucidum; SO, stratum oriens. B-E, Representative responses recorded from the CA3 pyramidal cell layer in response to hilar stimulation. Slices were from animals killed on the morning of proestrus (B), estrus (C), metestrus (D), or after ovariectomy (Ovx; E). For B-E, the top trace shows the first pair of responses to a pair of identical hilar stimuli (interstimulus interval, 40 msec). The next traces show responses selected from a stimulus train in which pairs of stimuli were triggered at 1 Hz for 5-10 sec. Responses are shown for the 1st, 3rd, 5th, or 10th pairs. Multiple population spikes are marked by arrows in B.

Figure 1.

Changes in BDNF immunoreactivity during the estrous cycle relative to ovariectomized and intact male rats. BDNF immunoreactivity is shown for sections of the following animals: A, proestrous female (Pro); B, estrous female (Est); C, metestrous female (Met); D, ovariectomized female (Ovx); E, intact male (Male). The immunoreactivity of mossy fibers (MFs; A, arrows) is strong at proestrus and estrus. Scale bar: (in A), 200 μm.

Figure 2.

Differences in BDNF immunoreactivity between proestrous, estrous, and metestrous rats. A, B, Sections from animals that were killed on the morning of proestrus (A) and estrus (B). Note that in A1 and A2, BDNF immunoreactivity is present in the granule cell layer (GCL), visible in A2 as multiple juxtaposed halos, presumably representing immunoreactive granule cell cytoplasm surrounding BDNF-negative nuclei. MOL, Molecular layer. In B1 and B2, immunoreactivity is weak in the granule cell layer relative to A1 and A2 but stronger in the hilus. This could reflect a shift in the distribution of BDNF within granule cells from a somatic location at proestrus to a primarily axonal distribution at estrus. Scale bars: (in A1) A1, B1, 150 μm; (in A2) A2, B2, 100 μm. C, A section from a metestrous rat that was processed concurrently with the sections shown in A and B. Mossy fiber immunoreactivity appears weak relative to A or B. Scale is the same as that in A1. D, An area of the pyramidal cell layer (PCL) of CA3b from an animal at proestrus shows immunoreactivity in stratum lucidum, the layer containing the mossy fibers (MFs). Some pyramidal cells (arrows) in the pyramidal cell layer are also immunoreactive. Scale bar: (in A2) 50 μm.

Figure 5.

Afferent specificity of area CA3 hyperexcitability. A, Recordings in the area CA3b pyramidal cell layer to the first and third pair of stimuli triggered 1 sec apart. The interstimulus interval between each pair was 40 msec. The slice was from an animal killed on the morning of estrus. B, Same recording site as A, but stimuli were triggered by fimbria stimulation and continued longer than A (for 10 sec). The responses to the 1st and 10th pairs of stimuli are shown. Unlike in A, multiple population spikes (A, arrows) did not occur in response to fimbria stimulation.

Figure 6.

Laminar specificity of area CA3 hyperexcitability at proestrus and estrus. A, Responses to the same hilar stimulus were recorded consecutively by moving the recording electrode to different strata of area CA3b. Recordings were from a proestrous rat and sites were positioned as shown by the x in Figure 4A. B, Simultaneous recordings from stratum pyramidale (SP) and stratum lucidum (SL) of CA3b in a slice from an estrous rat. The top two traces are the first pair of responses to hilar stimuli, recorded simultaneously in SP and SL. The bottom two traces are the responses to the third pair of stimuli. Pairs were triggered at 1 Hz. Arrows point to a spontaneous population spike and field EPSP. C, Sequential recordings in the same slice, using the same hilar stimuli. Only the third response of a train of three pairs of stimuli (each pair triggered 1 sec apart) is shown. SR, Recording in stratum radiatum; SL, subsequent recording in stratum lucidum; SP, subsequent recording in stratum pyramidale; SO, subsequent recording in stratum oriens. The largest field EPSP evoked by a stimulus, as well as the largest field EPSP recorded spontaneously, were always recorded in stratum lucidum, the layer corresponding to the mossy fibers. Note that the events occurring long after the pair of stimuli (arrows), indicative of hyperexcitability, had variable latencies. This was a characteristic of other experiments also.

Figure 7.

CA3 hyperexcitability is not antagonized by the NMDA receptor antagonist d-APV. A, Responses to a pair of hilar stimuli were recorded in stratum lucidum. Five pairs of stimuli were triggered at 1 Hz. The responses to the first and the fifth pairs are shown. Abnormal activity is marked by the arrows, indicative of hyperexcitability. B, After a 15 min exposure to 50 μm d-APV, the same stimuli shown in A still produced hyperexcitability.

Figure 8.

RIA analysis of reproductive hormones during the estrous cycle. Means ± SEM are shown for all cycle stages (Pro, proestrus; Est, estrus; Met, metestrus; Di2, diestrus 2), with the number of individual measurements indicated at the foot of each histogram bar. Significance of differences relative to hormone levels at estrus: ^*p < 0.05; ^**p < 0.01 (Scheffe's test).

Figure 9.

Effects of BDNF in area CA3 of a slice from a metestrous rat. A, In a slice from an animal killed on the morning of metestrus, the first and fifth responses to consecutive pairs of hilar stimuli at 1 Hz are shown. Only one population spike was evoked per stimulus (normal excitability). B, The responses to the same stimuli shown in A were triggered 60 min after adding BDNF (100 ng/ml) to the perfusing buffer. Multiple population spikes (arrows) were evoked in response to the fifth stimulus.

Figure 10.

Pharmacological reversal of hyperexcitability in area CA3 by K252a. A, Responses recorded in the pyramidal cell layer of area CA3 to paired hilar stimuli are shown. Before bath application of K252a, multiple population spikes occurred after five paired stimuli at 1 Hz (arrows). B, After K252a perfusion for 60 min, the same stimuli shown in A only evoked one population spike per stimulus.

Figure 11.

Pharmacological reversal of hyperexcitability in area CA3 by MLA. A, Responses recorded in the pyramidal cell layer of area CA3 in response to 1 Hz paired hilar stimuli are shown. Before bath application of MLA, multiple population spikes occurred after five paired stimuli at 1 Hz (arrows). B, After exposure to MLA for 20 min, the same stimuli shown in A only evoked one population spike per stimulus.

Figure 12.

Hypothetical mechanism for hyperexcitability in area CA3 at periovulatory stages of the estrous cycle. A, It is hypothesized that trkB receptors (trkBR) and α7nAChRs are present on mossy fiber terminals and that they modulate release of glutamate. AMPAR, AMPA receptor. B, At proestrus and estrus, the rise in estradiol triggers an increase in mossy fiber BDNF, so mossy fiber stimulation releases more BDNF and trkB receptor activation increases. TrkB receptors trigger a physical change in α7nAChRs, such as clustering of receptors, that in turn enhance α7nAChR function. An increase in glutamate release occurs as a result. Additional considerations are elaborated in Discussion.