The dentate gyrus filters incoming activity into the hippocampus proper. It plays a role in learning and memory and in pathological states such as epilepsy. Some of hilar interneurons of the dentate gyrus express neuropeptide Y (NPY), which modulates granule cell activity. A subpopulation of the NPY-expressing inhibitory interneurons is sensitive to seizure-induced damage. Pretreatment with β-estradiol in ovariectomized rats protects hilar interneurons against seizure-induced injury, including the NPY-containing damage-sensitive subpopulation. Here, we demonstrate that β-estradiol enhances NPY expression within the hilar interneurons. In vitro paired-pulse stimulation of the mixed perforant path revealed β-estradiol-induced augmentation of granule cell network inhibition, which at interstimulus intervals between 200 and 300 ms (corresponding to ∼3–5 Hz) was NPY sensitive and involved Y1 receptors, whereas it was insensitive to GABAB or metabotropic glutamate receptor antagonists. Additionally, β-estradiol pretreatment attenuated propagation of low-frequency (3.3 or 5 Hz) burst activity through the dentate gyrus. Scavenging endogenous NPY by intracerebroventricular administration of anti-NPY antibody accelerated kainic acid-induced seizure onset and increased seizure-induced neuronal damage in the hilus compared with rats treated with β-estradiol alone. Together, we show that β-estradiol upregulates hilar NPY and that this leads to enhancement in dentate gyrus inhibition of incoming frequencies between 3 and 5 Hz. Such frequencies are similar to the discharge frequencies recorded during seizure initiation in some patients with epilepsy. Thus, β-estradiol-induced NPY-sensitive filtering of 3–5 Hz frequencies may be an important regulator of incoming seizure activity, but it could also serve a physiological purpose in modulating information flow into the hippocampus proper.
Estrogens regulate gene expression and play an important role in modulation of neuronal activity. The effects of estrogens are mediated mainly by β-estradiol. The hippocampus represents an important target for β-estradiol, with most studies performed in the CA1 region (McEwen, 2002). There is little information on estrogen effects in regulation of neuronal excitability in the hippocampal dentate gyrus (Velíšková, 2007). This region is involved in complex biological functions, such as learning and memory consolidation (Pastalkova et al., 2006; Tashiro et al., 2006), and in pathological states, such as epilepsy (Lothman et al., 1992; Behr et al., 1998). It has been proposed that the dentate gyrus may serve as a gate for activity propagation into the hippocampus (Lothman et al., 1992). Dentate granule cell excitability, which is regulated by hilar inhibitory interneurons (Buckmaster and Schwartzkroin, 1995; Ratzliff et al., 2004), seems to determine ability of the dentate gyrus to filter incoming activity. Because hilar interneurons express estrogen receptors (Milner et al., 2001; Kalita et al., 2005), they may be a target of estrogen action.
Under physiological conditions, neuropeptide Y (NPY) is expressed in hilar interneurons. NPY regulates neuronal excitability by activating NPY-specific G-protein coupled receptors (Sperk et al., 1992; Baraban and Tallent, 2004). NPY is involved in modulation of physiological functions, including learning, memory, and anxiety, and in pathological states such as seizures (Vezzani et al., 1999; Heilig, 2004). A subpopulation of NPY-expressing hilar interneurons is especially vulnerable to seizure-induced injury (de Lanerolle et al., 1989). Loss of inhibitory interneurons contributes to increased dentate granule cell excitability (Martin and Sloviter, 2001) and may disrupt the ability of the dentate gyrus to filter incoming seizure activity (Heinemann et al., 1992). When the activity can propagate into hippocampus, it may lead to neuronal injury and intractable seizures (Engel et al., 1997; Mathern et al., 1997).
Chronic pretreatment with low doses (2 μg/d per rat) of β-estradiol prevents the loss of hilar neurons, including the NPY-expressing interneurons and the associated increase in granule cell excitability after prolonged seizures (status epilepticus) (Velíšek and Velíšková, 2002; Velíšková, 2006). The same dose of β-estradiol also delays the onset of kainic acid-induced seizures (Velíšková et al., 2000). Regulation of NPY expression by β-estradiol has been observed in other brain regions (Titolo et al., 2006), including the hippocampal CA1 field (Nakamura and McEwen, 2005), but such interaction has not been studied in the dentate gyrus. NPY upregulation protects against damage of hilar neurons (El Bahh et al., 2001) and delays seizure onset (Richichi et al., 2004; El Bahh et al., 2005). Given these positive and parallel effects of β-estradiol or NPY pretreatment on seizure-induced damage and on seizure susceptibility, we investigated β-estradiol–NPY interactions in the dentate gyrus. We determined β-estradiol-induced changes in NPY expression using immunohistochemistry and stereological counting. We studied synergistic influences of β-estradiol–NPY interactions in regulation of dentate gyrus network excitability using in vitro recordings. Finally, we used an in vivo approach to confirm that β-estradiol–NPY interactions are implicated in seizure susceptibility and protection of hilar neurons against seizure-induced damage.
Materials and Methods
Experiments were performed according to the Revised Guide for the Care and Use of Laboratory Animals and approved by the Albert Einstein College of Medicine Animal Care and Use Committee. Adult female Sprague Dawley rats (150–175 g; Taconic Farms, Germantown, NY) were used and kept on a 12 h light/dark cycle (lights on at 7:00 A.M.) with food and water ad libitum. All rats were ovariectomized (OVX) under ether anesthesia. After 7 d, rats were injected subcutaneously with either 17β-estradiol benzoate (β-estradiol; 2 μg/0.1 ml; Sigma, St. Louis, MO) or oil (0.1 ml; controls) for 4 consecutive days. Additional rats received either a single β-estradiol or oil injection.
Rats were perfused transcardially with 4% formaldehyde [four doses (4×) oil, n = 5; 4× β-estradiol, n = 4]. Brains were removed from the skull and postfixed in the same fixative overnight, cryoprotected with a 30% sucrose solution in PBS, pH 7.4, and frozen on dry ice. Staining was performed on free-floating coronal sections (40 μm) using our standard protocol for immunohistochemistry with the avidin–biotin peroxidase method (Ravizza et al., 2002). We used rabbit anti-NPY polyclonal antibody (1:2000; Sigma). NPY-immunoreactive neurons were visualized with 3′-3′-diaminobenzidine using the avidin–biotin horseradish peroxidase method (Vectastain ABC kit; Vector Laboratories, Burlingame, CA). Sections were then mounted on gelatinized slides, dehydrated, and coverslipped with Permount (Fisher Scientific, Pittsburgh, PA). To minimize differences between the various sets of immunohistochemistry, all sections were treated exactly the same way at all steps.
Control sections were prepared using the primary antiserum preabsorbed with the peptide or by incubating the sections without the primary antibodies: under both conditions, the immunohistochemical procedure failed to show any immunoreactivity.
Two independent observers, one of them blind to experimental conditions, performed cell counts. All sections throughout the hippocampus were collected, counted, and filed. The stereological counting was performed according to the West method (West, 2002). Our areas of interests were the hilus (defined as the area surrounded by the layer of dentate granule cells and an imaginary connection between the ends of both granule cell blades except for interposed layer of pyramidal neurons of CA3) and CA1 hippocampal region. Only the dorsal hippocampus has been examined [between the level of bregma −2.12 and −4.52 mm (Paxinos and Watson, 1998)], and, for the in vitro recordings, slices have also been cut from the dorsal hippocampus. Every sixth section that contained the area of interest was chosen for examination after selecting randomly the first one within the first interval (12 sections for each animal). This was the section sampling fraction (ssf). All NPY-positive neurons with intact morphology, which came to focus within the dissector height, were counted in each area of interest. The optical dissector height (h) relates to a known fraction of section thickness (t). Height of the optical dissector was 10 μm. Then, the estimated number of neurons is ΣQ × 1/ssf × t/h, in which ΣQ is the total number of neurons counted in the region of interest. The data were compared between the groups.
In vivo anti-NPY antibody treatment. Six days after the ovariectomy (4× oil, n = 13; 4× β-estradiol, n = 13), unilateral stainless steel cannula was stereotaxically implanted into the third ventricle (anteroposterior, −3 mm; lateral, 0.1 mm; depth, −4.5 mm; incisor bar at −3.5 mm) under combined ketamine (70 mg/kg) and xylazine (7 mg/kg) anesthesia administered intraperitoneally. Mini-osmotic pumps (ALZET model 1002; ALZA, Mountain View, CA) were placed between the shoulder blades and connected to the cannula via a catheter. ALZET pumps were filled with the anti-NPY antibody (1:5) or normal rabbit serum (NRS) (1:3). This concentration of antibody has been shown to accelerate the kindling rate with no influence on spontaneous behavior or the background EEG activity (Reibel et al., 2003). The dilution of both agents was adjusted to ensure similar protein content of ∼10 μg/μl. The anti-NPY antibody (Sigma) was developed in rabbit using synthetic porcine NPY conjugated to keyhole limpet hemocyanin as immunogen. The pump delivered a continuous flow of solution at a rate of 0.25 μl/h. The anti-NPY treatment started 1 d before β-estradiol treatment and continued until the end of the experiment. The spread of the anti-NPY antibody was determined using immunohistochemistry omitting the primary antibody and detected as NPY-positive staining. The area, in which the antibody did not penetrate, was immunonegative.
Kainic acid-induced seizures.
Rats with the ALZET pump implants (see above) were injected with kainic acid (15 mg/kg, i.p.; Ocean Produce International, Shelburne, Nova Scotia, Canada) (Velíšková et al., 2000). The behavior was continuously registered using a video monitoring system for subsequent analysis of seizure severity. Only rats with continuous long-lasting behavioral seizures (status epilepticus lasting at least 4 h) were used. The status epilepticus was terminated by pentobarbital (50 mg/kg, i.p.) after 4 h. The following parameters were analyzed: latency to first forelimb clonus and latency to status epilepticus onset.
FluoroJade B staining.
This is a sensitive method for detecting dying neurons (Pitkanen et al., 2002). All rats were transcardially perfused 2 d after the status epilepticus. Coronal sections (30 μm) containing the dorsal hippocampus [between the level of bregma −2.12 and −4.52 mm (Paxinos and Watson, 1998)] were cut on a cryostat at −20°C. Every fifth section was collected (16 sections in each animal). Mounted sections were immersed in alcohol with 1% NaOH, transferred into 0.06% potassium permanganate, and then incubated with FluoroJade B (0.0004%; Histo-Chem, Jefferson, AR), followed by washing in 0.1 m PBS, dried, soaked in xylene, and coverslipped with Permount.
Brain slice preparation and paired-pulse paradigm.
Rats were decapitated under deep ether anesthesia 24 h after the last β-estradiol/oil injection. Horizontal slices (4× oil, n = 22; 4× β-estradiol, n = 22; 1× oil, n = 18; 1× β-estradiol, n = 8) containing the dorsal hippocampus (400 μm thick) were cut on a vibratome in ice-cold artificial CSF (ACSF) (in mm: 126 NaCl, 5 KCl, 1.25 NaH2PO4, 2 MgCl2, 2 CaCl2, 26 NaHCO3, and 10 glucose, pH 7.36). The slices were preincubated in an interface chamber in warmed (34–35°C) and oxygenated ACSF (5% CO2/95% O2) for at least 1 h. Paired square pulses (50–400 μA, 150 μs duration) were delivered via a stainless steel bipolar stimulating electrode (Frederick Haer Company, Bowdoinham, ME) positioned over the outer and middle third of the molecular layer (mixed perforant path stimulation) (Velíšek and Velíšková, 2002) using a stimulator coupled to a stimulus isolation unit (A.M.P.I., Jerusalem, Israel). Interstimulus intervals between the paired stimuli ranged from 10 to 1000 ms, corresponding to frequencies of 100 to 1 Hz. The paired stimuli were repeatedly applied to the same tissue every 30 s. This interval ensured full recovery between stimuli so that the response to one stimulus pair was not affected by the previous stimulus pair (Haas et al., 1996). Extracellular evoked responses (population spikes) were recorded using glass recording micropipette (1–5 MΩ) filled with 2 m NaCl placed in the middle third of the external blade of the dentate gyrus granule cell layer (stratum granulosum). We preferred to record extracellular network (population) responses because β-estradiol may have differential effects in individual cells. Transmission of perforant path stimulation through granule cells, i.e., dentate gating, was quantified by measuring population spike amplitude. Data were digitized and computer analyzed using Axoscope software (Molecular Devices, Sunnyvale, CA). For each interstimulus interval, 6–10 paired responses were averaged. After an initial cycle of control measurements consisting of 6–10 paired-pulse stimulations at each interstimulus interval between 10 and 1000 ms, test substances were bath introduced to the chamber, followed by a cycle of paired-pulse measurements at each interstimulus interval.
In vitro treatments. To block NPY availability, anti-NPY antibody (1:1000; Sigma) was bath applied for at least 1 h before recordings in slices (4×, n = 12; 4× β-estradiol, n = 11). After each use of the anti-NPY antibody, the tubing and the recording chamber were flushed for at least 10 min with the ACSF containing bovine serum albumin at 1:1000 to remove remnant anti-NPY antibody, followed by ACSF and then by distilled water for at least 1 h. Control experiments included recordings in slices from β-estradiol-treated OVX rats (n = 9) perfused with the ACSF containing preimmune rabbit serum [1:1000 for 1 h (Sigma); rabbit was the host for the production of anti-NPY antibody], as well as recordings in slices from OVX oil controls with bath application of anti-NPY antibody (n = 12) or NPY [n = 7 (Sigma); 1 μm for 1 h (Klapstein and Colmers, 1997)]. Similar cleaning procedures for the tubing and the chamber were used as described above. To determine the role of Y1, Y2, and Y5 NPY receptors, the following specific antagonists to individual NPY receptors were bath applied for at least 30 min before recording: 100 nm 6-[[2-(1-methylethyl)phenyl]]sulfonyl-5-nitro-8-quinolinamine (PD 160170) (Wielgosz-Collin et al., 2002) (Y1 receptor antagonist; n = 7), 30 or 100 nm (S)-N2-[[1-[2-[4-[(R,S)-5,11-dihydro-6(6h)-oxodibenz[b,e]azepin-11-yl]-1-piperazinyl]-2-oxoethyl]cyclopentyl]acetyl]-N-[2-[1,2-dihydro-3,5[4H)-dioxo-1,2-diphyenl-3H-1,2,4-triazol-4-yl]ethyl]argininamide (BIIE 0246) (El Bahh et al., 2002; Tu et al., 2005) (Y2 receptor antagonist; 30 nm, n = 8; 100 nm, n = 6), and 250 nm 5,5-dimethyl-2-(2,3,4,9-tetrahydro-3,3-dimethyl-1 oxo-1H-xanthen-9-yl)-1,3-cyclohexanedione (L-152,804) (Woldbye et al., 2005) (Y5 receptor antagonist; n = 5). To determine the role of GABAB receptors in the β-estradiol-induced enhancement of the late paired-pulse inhibition, 400 μm (3-aminopropyl)(diethoxymethyl)phosphinic acid (CGP 35348) (Velíšek and Velíšková, 2002) was bath applied for at least 30 min before recordings (n = 10). LY 341495 [(2S)-2-amino-2-[(1S,2S)-2-carboxycycloprop-1-yl]-3-(xanth-9-yl) propanoic acid] was bath applied for at least 30 min before recordings at 20 μm (n = 8) or 200 nm (n = 7) to determine the role of metabotropic glutamate receptors (mGluRs) in the effects of β-estradiol pretreatment. In micromolar concentrations, LY 341495 is a broad-spectrum mGluR antagonist (Fitzjohn et al., 1998). At nanomolar concentrations, it is a selective group II mGluR antagonist (Fitzjohn et al., 1998). All antagonists to NPY, GABAB, and metabotropic glutamate receptors were purchased from Tocris Bioscience (Ellisville, MO).
An individual slice was used for recording of a control paired-pulse cycle plus one additional cycle testing one of the listed treatments. We used a maximum of two slices from one rat for any of the conditions.
Burst activity propagation.
This experiment was designed to confirm that the β-estradiol treatment decreases propagation of burst activity through the dentate gyrus at frequencies identified in the paired-pulse paradigm as NPY sensitive (3.3–5 Hz). Brain slices containing the dorsal hippocampus from 4× β-estradiol-treated (n = 7) or 4× oil-injected rats (n = 7) were prepared as above. Mixed perforant path was stimulated with a burst of six rectangular monophasic pulses with the intensity at 70% of the current eliciting maximal response in the granule cell layer. We tested responses of granule cell network to bursts of low-frequency stimulation at frequencies of 3.3 and 5 Hz corresponding to the NPY-sensitive paired-pulse intervals (300 and 200 ms, respectively) and to an arbitrarily chosen control high-frequency stimulation at a frequency of 20 Hz, which corresponded to an NPY-insensitive paired-pulse interstimulus interval (50 ms). We evaluated the ratio of the sixth response to the first to determine the transmission of burst activity through the dentate gyrus. Results were compared between β-estradiol-treated and oil-injected rats.
In every statistical evaluation presented here, tests for normality and variance were run on the data. Because we consistently found Gaussian distribution and similar variances among the groups compared in the individual tests, parametric statistics was used.
Latency to onset of kainic acid-induced clonic seizures was compared using one-way ANOVA (factor of treatment) followed by pairwise post hoc Fisher's protected least significance difference test. Cell counts were compared between the two conditions (oil vs β-estradiol pretreatment) using two-tailed Student's t test. The ratio of the second (test) population spike amplitude to the amplitude of the first (conditioning) population spike in the response averaged from 6–10 stimulations also yielded normally distributed values with similar variances across different groups. For the initial evaluation comparing test and control conditions, we used ANOVA. If differences were significant, we used Duncan's multiple range t test to compare interstimulus interval-matched data (Derbenev et al., 2006). Every individual data subset entered post hoc analysis only once. Level of significance was always preset to p < 0.05.
β-Estradiol increases NPY expression within the hilar interneurons
Consistent with a preliminary report in an abstract form (Velíšková, 2003), NPY immunoreactivity was detected in significantly higher number of neurons in the hilar region of β-estradiol-treated rats (n = 4) compared with OVX controls (n = 5) that received oil (vehicle) injections (β-estradiol, 41.16 ± 5.5 × 103; oil, 22.51 ± 3.17 × 103; Student's t test, *p < 0.05). The NPY-positive neurons in β-estradiol-treated rats appeared darker than the ones in oil-injected rats (Fig. 1). It should be noted here that the immunohistochemistry was always performed in parallel on slices from β-estradiol-treated and oil-injected animals, and the individual steps of the procedure were strictly the same to allow comparisons between the treatments. Treated rats received four daily injections of 2 μg/rat β-estradiol, which produces plasma levels of β-estradiol within the physiological range (Neal-Perry et al., 2005). Because a recent study focusing specifically on the CA1 region showed enhanced NPY mRNA expression as well as increased number of NPY-immunoreactive neurons after 10 μg/rat β-estradiol (Nakamura and McEwen, 2005), we also determined changes in the NPY expression in CA1. The 2 μg/rat dose of β-estradiol used in the present study did not increase the number of NPY-expressing neurons in the CA1 region compared with oil controls (Student's t test, p = 0.80). In addition, there were no such apparent differences in the immunointensity between β-estradiol-treated and oil-injected animals in the CA1 region as we observed in the hilus.
β-Estradiol-mediated increase in hilar NPY plays a role in kainic acid-induced seizures
Because enhanced expression of NPY in the hilar region is associated with powerful anticonvulsant and neuroprotective actions (Richichi et al., 2004; Dube et al., 2005; Silva et al., 2005), we tested whether β-estradiol-induced delay in seizure onset and neuroprotective effects were mediated by an increase in NPY. We reduced NPY activity by intracerebroventricular administration of anti-NPY antibody to scavenge the endogenous NPY. The presence of anti-NPY antibody in the hippocampal hilar region in β-estradiol-treated OVX rats (n = 7) blocked the β-estradiol-induced (n = 6) increases in seizure latency. Seizure latency in anti-NPY β-estradiol-treated rats was similar to OVX oil (n = 7) controls (ANOVA; F(2,17) = 13.15; *p < 0.001) (Fig. 2A), in which ovariectomy was associated with low NPY expression (Fig. 1). To confirm that the effect on seizure latency was not attributable to surgery and subsequent intracerebroventricular injection-related stress, we compared the seizure latency in the β-estradiol-treated OVX rats with intracerebroventricular administration of preimmune (normal) rabbit serum (n = 4) to the β-estradiol-treated OVX rats without any intracranial implants (n = 6). Seizure latency was not affected by surgery or subsequent intracerebroventricular administration of normal rabbit serum compared with rats without surgery (Student's t test, p = 0.76). As we reported previously (Velíšková et al., 2000), β-estradiol pretreatment did not prevent development of status epilepticus. In addition, consistent with previous studies, behavioral seizure severity was not affected by β-estradiol pretreatment (Velíšková et al., 2000; Reibel et al., 2000; Hoffman et al., 2003). Finally, anti-NPY antibody pretreatment also abolished the neuroprotective effect of β-estradiol in the hilar region as determined by FluoroJade B staining (Fig. 2B–D).
β-Estradiol enhances the late paired-pulse inhibition in the dentate gyrus
We used the in vitro paired-pulse paradigm by stimulating the mixed perforant pathway to assess changes in the dentate granule cell network excitability (Velíšek and Velíšková, 2002). The paired-pulse curve was constructed by calculating population spike amplitude ratio (R2/R1) at increasing interstimulus intervals. The curve consisted of three components (Haas et al., 1996; Velíšek and Velíšková, 2002) referred to here as (1) early inhibition (10–50 ms), (2) intermediate facilitation (70–125 ms), and (3) late inhibition (150–1000 ms). Slices from OVX females treated with four daily doses of β-estradiol (2 μg/rat; n = 22) displayed significant enhancement of late paired-pulse inhibition at interstimulus intervals 125–1000 ms compared with slices from OVX controls (n = 22) receiving oil (two-way ANOVA; main effect of treatment, F(1,42) = 15.871; *p < 0.001). The post hoc comparisons revealed significant differences in the following paired-pulse intervals: *p125–150ms < 0.01; *p200–1000ms < 0.001 (Fig. 3A). There were no changes in paired-pulse paradigm in the interstimulus intervals of 10–100 ms (p10–100ms > 0.05).
We also tested the effect of a single β-estradiol (2 μg) dose on granule cell excitability. It is possible that a single dose might increase network excitability, whereas four β-estradiol injections could elicit a slower, compensatory increase in the network inhibition. Similar phenomenon has been described in the CA1 region after 10 μg of β-estradiol (Rudick and Woolley, 2001). This study showed that inhibitory synaptic input to pyramidal neurons in CA1 decreases after a single β-estradiol injection, but it increases after two β-estradiol injections (Rudick and Woolley, 2001).
We found that a single β-estradiol injection had no effect on paired-pulse profiles (two-way ANOVA; main effect of treatment, F(1,24) = 0.998; p > 0.05; 1× β-estradiol, n = 8; 1× oil OVX, n = 18) (Fig. 3B), suggesting that β-estradiol does not alter the dentate gyrus network excitability after just one injection.
NPY is involved in β-estradiol-induced enhancement of paired-pulse inhibition at interstimulus intervals between 200 and 300 ms
We used bath application of anti-NPY antibody to scavenge the endogenous NPY. In slices from OVX rats pretreated with four doses of β-estradiol (n = 11), addition of the anti-NPY antibody (1:1000) to the ACSF induced a significant loss of paired-pulse inhibition at interstimulus intervals between 200 and 300 ms (paired t test; *p200–300ms < 0.05) (Fig. 4A) but did not significantly influence earlier or later intervals (p125–150ms > 0.05; p500–1000ms > 0.05). Preimmune normal rabbit serum (1:1000) bath applied to slices from β-estradiol-treated rats (n = 9) for at least 1 h had no effect on the paired-pulse responses affected by the anti-NPY antibody (paired t test, p200–300ms > 0.05) (Fig. 4B). To confirm the specificity of this effect, NPY was bath applied onto slices from OVX oil-injected rats, which have low NPY levels (Fig. 1). In slices from OVX oil controls (n = 7), perfusion with 1 μm NPY enhanced the late paired-pulse inhibition selectively and specifically at the interstimulus intervals between 200 and 300 ms (paired t test, *p200–300ms < 0.05) (Fig. 4C). Conversely, bath application of anti-NPY antibody in slices from OVX oil controls (n = 12) had no effect on the paired-pulse responses (paired t test; p200–300ms > 0.05).
NPY mediates its inhibitory effects in the dentate gyrus of the dorsal hippocampus by inhibition of glutamate release via activating presynaptic Y1 or Y2 receptors (Silva et al., 2001). Because the dorsal hippocampus was the region of our interest, we mainly concentrated on determining the involvement of Y1 and Y2 NPY receptor subtypes. Bath application of the NPY Y1 receptor antagonist PD 160170 (100 nm; n = 7) blocked the β-estradiol-induced enhancement of the late paired-pulse inhibition at interstimulus intervals of 150 ms and between 200 and 300 ms (paired t test, *p150ms < 0.05 and *p200–300ms < 0.01) (Fig. 4D), suggesting involvement of this NPY receptor subtype. Conversely, bath application of the NPY Y2 receptor antagonist BIIE 0246 (30 nm, n = 8; or 100 nm, n = 6) had no effect (paired t test, p125–1000ms > 0.05) (Fig. 4E). Finally, activation of NPY Y5 receptors may mediate inhibitory effects in the hippocampus as well (Woldbye et al., 2005), although another study did not confirm inhibitory effects of Y5 receptors in the hippocampus (El Bahh et al., 2005). Also, binding studies show that Y5 receptor subtype is preferentially expressed in the ventral hippocampus (Dumont et al., 1998). Accordingly, in our preparation from the dorsal hippocampus, bath application of NPY Y5 receptor antagonist L-152,804 (250 nm, n = 5) did not influence the β-estradiol-induced effects in the late paired-pulse intervals (paired t test, p125–1000ms > 0.05) (Fig. 4F).
We performed additional experiments to confirm that the effect at the paired-pulse intervals between 200 and 300 ms is specific for the NPY system. We tested the involvement of the GABAB and the mGluR-mediated transmission, which represents the slow inhibition via a second messenger and thus possibly involved in the late phase of paired-pulse paradigm. Bath application of 400 μm of the GABAB receptor antagonist CGP 35348 (n = 10) for at least 30 min before recordings did not have any effect on the late paired-pulse inhibition (two-way ANOVA, F(1,18) = 0.07; p125–1000ms > 0.05) (Fig. 5A).
To test the mGluR system, we used bath application of 20 μm LY 341495, which at this concentration is a broad-spectrum mGluR antagonist (Fitzjohn et al., 1998). In slices from β-estradiol-treated rats (n = 8), 20 μm LY 341495 did not influence the NPY-mediated increase in the inhibition at the paired-pulse intervals between 200 and 300 ms but selectively suppressed the β-estradiol-induced enhancement of paired-pulse inhibition at the very late (500–1000 ms) interstimulus intervals (paired t test, *p500–1000ms < 0.05) (Fig. 5B). It is possible that, by blocking all three types of mGluR, the lack of effect may be attributable to antagonizing effects between the excitatory type I mGluRs and the inhibitory type II mGluRs. Thus, we also bath applied LY 341495 using the dose of 200 nm (n = 10). At this dose, LY 341495 is a specific group II mGluR antagonist (Fitzjohn et al., 1998). At this concentration, LY 341495 again did not influence the paired-pulse intervals between 200 and 300 ms but suppressed the β-estradiol-induced paired-pulse inhibition at 150 ms interstimulus interval (paired t test, *p150ms < 0.05) (Fig. 5C).
β-Estradiol-treatment attenuates the propagation of burst activity through the dentate gyrus
To verify that 4× β-estradiol pretreatment may attenuate the propagation of bursting activity through the dentate gyrus at the NPY-sensitive frequencies identified with the paired-pulse paradigm, we used the in vitro stimulation of mixed perforant path with bursts of six pulses delivered either at NPY-sensitive frequencies (3.3 or 5 Hz; corresponding to interstimulus interval 300 or 200 ms, respectively) or at the NPY-insensitive frequency (20 Hz; corresponding to interstimulus interval 50 ms). Propagation of the low-frequency (3.3 or 5 Hz) burst activity (expressed as R6/R1) through the dentate gyrus was decreased in slices from β-estradiol-treated rats compared with slices from oil-injected OVX controls (paired t test, *p < 0.05) (Fig. 6B). Propagation of the high-frequency stimulation (20 Hz) was not different between slices from β-estradiol-treated and oil-injected rats (paired t test, p > 0.05) (Fig. 6B). These data together with the results of the paired-pulse paradigm indicate that the dentate gyrus in β-estradiol-treated rats can better filter the incoming low-frequency activities at ∼3–5 Hz (NPY-sensitive) than the dentate gyrus in oil-injected rats. The filtering of high-frequency activities is not affected by β-estradiol.
Our study has identified several novel mechanisms of β-estradiol-mediated effects on neuronal excitability in the dentate gyrus. First, the 4-d β-estradiol treatment in OVX female rats using a dose that produces estradiol plasma levels within a physiological range increases the number of NPY-immunopositive cells in the dentate hilar region compared with oil controls. Second, this NPY increase is associated with specific enhancement in late phase of paired-pulse inhibition (at interstimulus intervals between 200 and 300 ms) in the perforant path–dentate gyrus system determined in vitro and involves activation of NPY Y1 receptors. This effect further extends into increased filtering of burst discharges between ∼3 and 5 Hz incoming to the dentate gyrus via the mixed perforant path. Third, β-estradiol-induced increase in NPY seems to be responsible for delayed onset of kainic acid-induced seizures and neuroprotection against status epilepticus-induced hilar damage because both effects can be blocked by intracerebroventricular administration of anti-NPY antibody. Results from the present study establish a link between the effects of β-estradiol and NPY and a mechanism to modulate dentate gyrus function.
Repeated administration of a physiological dose of β-estradiol in OVX females leads to upregulation of NPY in the hilus compared with oil-injected controls, which have a low NPY expression. We speculate that the increased number of immunopositive neurons in the hilus after β-estradiol treatment may be attributable to enhanced expression of the peptide within the NPY-expressing neurons but not attributable to de novo production of NPY. Overexpression of NPY in the hilus delays seizure onset and has neuroprotective effects (El Bahh et al., 1997; Vezzani and Sperk, 2004). NPY overexpression can be also associated with reduced seizure duration/severity and thus may lead to a decrease of neuronal damage (Richichi et al., 2004; Dube et al., 2005). However, we did not observe a decrease in seizure severity in our study. This finding suggests a possible dissociation between neuroprotective effects of NPY and reduction of seizure severity.
The hilar region-specific effect on NPY expression may be associated with the treatment paradigm of 2 μg/rat β-estradiol used in this study, whereas a higher dose (10 μg/rat) has been shown to increase NPY expression in the CA1 region (Nakamura and McEwen, 2005). Such biphasic effect is not surprising because administration of a 10 μg dose of β-estradiol enhances sensitivity to NMDA receptor-mediated transmission in field CA1 (Woolley et al., 1997), an effect not present after administration of the 2 μg dose of β-estradiol (Velíšek et al., 1999).
The β-estradiol-induced increase in NPY expression in hilar interneurons leads to trans-synaptic regulation of granule cell activity. Hilar NPY interneurons have axons ramifying in the perforant path terminal field and thus participate in regulation of this major excitatory input to the hippocampus proper (Freund and Buzsaki, 1996). Because we directly stimulated the mixed perforant pathway (in the molecular layer of the dentate gyrus), we do not expect any influence of potential β-estradiol-induced changes occurring upstream the stimulation site (such as in the entorhinal cortex) because our stimulating/recording paradigm bypasses these influences. The β-estradiol-induced increase in NPY expression in the hilar region was associated with enhanced dentate gyrus filtering at interstimulus intervals corresponding approximately to the frequencies between 3 and 5 Hz. Indeed, if bursts of stimuli were delivered at these frequencies, the responses of granule cells in β-estradiol-treated rats were attenuated compared with responses in oil-injected rats. Increased synaptic inhibition at these specific frequencies may be important for filtering of kainic acid-induced epileptiform activity originating in the entorhinal cortex (Ben-Ari et al., 1981), which involves increases in cortical neuronal activity at frequencies of 2–5 Hz (Medvedev et al., 2000). Also in patients with temporal lobe epilepsy with hypersynchronous onset of seizures, deep electrode recordings from the hippocampus show increase in frequency of interictal spikes with spike-and-wave shape (corresponding to frequency of 3 Hz) during the very initial stage of a seizure (Engel, 1989). Thus, enhanced dentate filtering of these frequencies might disrupt seizure propagation. Accordingly, in vivo, we found a delay in kainic acid-induced seizure onset and neuroprotection of the hippocampal neurons. Both effects were abolished after in vivo NPY scavenging within the hilar region.
NPY is stored in dense-core vesicles and may require high-frequency stimulation for its release (Hokfelt, 1991). However, spontaneous or tonic NPY release has been reported in the hippocampus (Tu et al., 2005) and in the hypothalamus (Fu et al., 2004). We found that NPY participates in transmission determined by the paired-pulse paradigm in the β-estradiol-treated rats, suggesting that spontaneous release of increased NPY may account for the results here.
Our data show that the NPY-mediated enhancement of perforant path-granule cell network inhibition involves activation of NPY Y1 receptors. The Y1 receptor antagonist PD 160170 completely blocked the β-estradiol-induced inhibition enhancement at interstimulus intervals of 200–300 ms. Activation of presynaptic Y1 receptors in the dentate gyrus has been shown to suppress glutamate release (Silva et al., 2001), which is consistent with an inhibitory effect on neuronal activity. Conversely, blockade of Y2 receptors did not affect the late paired-pulse inhibition using our paired-pulse paradigm by stimulating the mixed perforant path, which suggests that the Y2 receptors may not be involved in regulation of neuronal activity at perforant path–granule cells synapses. This is in accordance with binding studies showing that Y2 receptors are mainly located in the hilar region, whereas the Y1 receptors are within the molecular layer (Gobbi et al., 1998). We also did not find any involvement of the Y5 receptors, which is most likely attributable to the fact that these receptors are expressed in ventral rather than dorsal hippocampus (Dumont et al., 1998).
No effect of β-estradiol was found on paired-pulse intervals between 10 and 100 ms. These intervals correspond to early inhibition and intermediate facilitation phases of the paired-pulse paradigm, which are sensitive to changes in fast neurotransmission. Thus, these results suggest that fast neurotransmission mediated via ionotropic receptor systems is not affected by the β-estradiol treatment paradigm. This finding is similar to the findings in the CA1 region reported by others (Smith and McMahon, 2005).
Because the last β-estradiol injection was 24 h before testing, the β-estradiol-induced effects seem to involve genomic action mediated by intracellular estrogen receptors (Gruber et al., 2002). The contribution of acute, membrane effects of β-estradiol is unlikely because the NPY-dependent increase in the paired-pulse inhibition could not be detected after a single β-estradiol injection.
The paired-pulse paradigm revealed that β-estradiol treatment also activated mGluRs. Because activation of groups II and III mGluR increases NPY expression in the hippocampus (Smialowska and Bajkowska, 1997; Schwarzer and Sperk, 1998), this raises a possibility that the β-estradiol-induced increase in NPY expression may be induced indirectly by activation of the mGluR system. However, a single mGluR-activating stimulus within 24 h before the NPY assay already effectively increased the NPY expression (Smialowska and Bajkowska, 1997; Schwarzer and Sperk, 1998). Acute application of β-estradiol on hippocampal cultures has been shown to activate mGluRs (Boulware et al., 2005). This effect seems to involve membrane estrogen receptors, because the mGluR activation occurs very rapidly (Boulware et al., 2005). Thus, one would expect to find the enhanced NPY-mediated inhibition already after a single β-estradiol injection as a result of the acute activation of mGluRs. However, there were no changes in the paired-pulse paradigm at any interstimulus interval after one β-estradiol injection. Finally, we found that, after blocking mGluRs by bath application of the mGluR antagonist LY 341495, the enhanced response at interstimulus intervals between 200 and 300 ms sensitive to the anti-NPY antibody is still present. Together, these results suggest that the β-estradiol-induced effects on the NPY and mGluR system may be independent from each other.
The dentate gyrus plays an important function in cognitive processes (Kirkby and Higgins, 1998; Pastalkova et al., 2006). The β-estradiol-induced increase in dentate gyrus filtering at selective frequencies may have an important role in these physiological tasks by improving information processing. The decline of hippocampal NPY attributable to the lack of β-estradiol may be involved in learning and memory disturbances and changes in mood, including increased anxiety states in women after menopause (medical or natural) (Hattiangady et al., 2005; Osterlund et al., 2005). These clinical observations are supported by animal studies, which also show that ovariectomy leads to decline in learning and memory and to increased anxiety in females, all of which can be corrected by β-estradiol replacement (El-Bakri et al., 2004; Imwalle et al., 2005). Similarly, changes in the NPY system are associated with disturbances in learning and memory as well as anxiety behaviors (Heilig, 2004; Redrobe et al., 2004). These parallel effects of β-estradiol and NPY may imply a common pathway of action and interactions between both systems. Thus, our data identify a mechanism mediating estrogen-induced regulation of hippocampal physiology. This mechanism may constitute a novel therapeutic target for the design of treatments to preserve or restore impaired hippocampal function.
This work was supported by National Institutes of Health Grants NS-41366 and NS-20253 and a Research Grant from CURE Foundation. We thank Drs. S. L. Moshé, P. K. Stanton, and F. A. Lado for constructive comments and scientific discussion during preparation of this manuscript and Dr. C. Hall for consultation in statistics.
- Correspondence should be addressed to Dr. Jana Velíšková, Albert Einstein College of Medicine, K 312, 1410 Pelham Parkway South, Bronx, NY 10461.