Local Estrogen Synthesis Regulates Parallel Fiber–Purkinje Cell Neurotransmission Within the Cerebellar Cortex

Local Estrogen Synthesis Regulates Parallel Fiber–Purkinje Cell Neurotransmission Within the... Abstract Estrogens affect cerebellar activity and cerebellum-based behaviors. Within the adult rodent cerebellum, the best-characterized action of estradiol is to enhance glutamatergic signaling. However, the mechanisms by which estradiol promotes glutamatergic neurotransmission remain unknown. Within the mouse cerebellum, we found that estrogen receptor activation of metabotropic glutamate receptor type 1a strongly enhances neurotransmission at the parallel fiber–Purkinje cell synapse. The blockade of local estrogen synthesis within the cerebellum results in a diminution of glutamatergic neurotransmission. Correspondingly, decreased estrogen availability via gonadectomy or blockade of aromatase activity negatively affects locomotor performance. These data indicate that locally derived, and not just gonad-derived, estrogens affect cerebellar physiology and function. In addition, estrogens were found to facilitate parallel fiber–Purkinje cell synaptic transmission in both sexes. As such, the actions of estradiol to support cerebellar neurotransmission and cerebellum-based behaviors might be fundamental to understanding the normal processing of activity within the cerebellar cortex. For decades, estrogens have been known to affect cerebellar function and development in both male and female animals (1–3). Early in rodent development, estrogens influence cerebellar synaptic connectivity and other aspects of its neuroanatomical organization (4–6). Within the adult, estrogens enhance synaptic efficacy and augment cerebellum-mediated behaviors (7–10). The clinical deficits occurring after the administration of estrogen receptor antagonists and aromatase inhibitors in humans also suggest a central role of estradiol in cerebellar function (11–16). Additionally, numerous studies have described neuroprotective effects of estrogens within this brain region (17–20). However, despite all this evidence, we lack a clear understanding regarding the mechanisms by which estrogens exert influence on the cerebellum. Recent findings have suggested that in addition to the activation of nuclear hormone receptors, estrogens affect nervous system function via multiple signaling pathways that are initiated at the cellular membrane (21, 22). Also, peripheral estrogens might not be the sole source of ligand, because evidence from both human and animal studies has shown that estradiol is synthesized locally within the cerebellum (4, 6, 23, 24). Through technological advancement of in vivo imaging studies (25, 26), in conjunction with both established electrophysiological and behavioral techniques, we have reexamined estrogen action within the rodent cerebellum. We have defined a mechanism by which estrogens support normal cerebellar function. In both males and females, estrogen receptor activation supports metabotropic glutamate receptor type 1a (mGluR1a) neurotransmission, which is required for proper parallel fiber (PF)–Purkinje cell synaptic function. Moreover, activation of cerebellar estrogen receptors is at least partially mediated by local estrogen synthesis. Consequently, estrogen depletion results in deficits in locomotor activity, demonstrating the physiological importance of this hormone for normal behavior. Materials and Methods Animals For the mouse experiments at the University of Minnesota, the Institutional Animal Care and Use Committee approved the procedures. Intact and gonadectomized mice (strain, Friend Virus B; age, 3 to 4 months) arrived from Charles River Laboratories (Wilmington, MA). The mice were housed in groups of four (separated by sex) in plastic cages and maintained in a specific pathogen-free colony room. The animal room was maintained at a controlled temperature with a 12-hour light/dark schedule. Food and water were available ad libitum. Experimental procedures began 1 week after acclimation. Experiments in rats were performed at Nanjing University and were in compliance with the U.S. National Institutes of Health Guide for the Care and Use of Laboratory Animals. Patch clamp experiments used male and female rats (Sprague-Dawley; Experimental Animal Center of Nanjing Medical University, Nanjing, China) aged 12 to 16 days. The younger age of the rats was necessary to obtain stable patch clamp recordings. The data are presented as the mean ± standard error of the mean throughout. DigiGait™ acquisition and analysis The mice were given a subcutaneous osmotic minipump implant under isoflurane anesthesia (1.5% to 2%). To prepare for the midscapular incision, the fur was removed, and the area was disinfected with betadine. A midscapular incision was made through the skin, and a subcutaneous pocket was created by opening and closing a hemostat between the skin and muscle and spreading the subcutaneous tissues apart. ALZET osmotic minipumps (model no. 2002; Durect Corporation, Cupertino, CA) were filled under sterile conditions with either the aromatase inhibitor fadrozole (Fad; catalog no. F3806; Sigma-Aldrich, Burlington, MA) in 0.9% saline (0.5 mg/kg/d) or 0.9% saline as per the manufacturer’s instructions. A filled osmotic minipump was inserted into the pocket, with the flow modulator of the pump pointing away from the incision. The incision was closed using wound clips. One week after surgery, the mice were tested for differences in locomotor function. The DigiGait™ System (Mouse Specifics, Inc., Framingham, MA) performs gait analysis of rodents over a range of walking and running speeds by monitoring an animal’s gait continuously via ventral plane images taken through a transparent motorized treadmill at a rate of 80 frames/s. These images generate digital paw prints and a dynamic gait signal for each of the four limbs and quantifies both spatial and temporal indexes of gait. The mice were tested at a “running” speed of 25 cm/s. Videos (5 to 7 seconds) of each mouse were analyzed using the DigiGait™ software, version 12.4, to automatically calculate the values for the gait parameters. The effects were analyzed through two-way analysis of variance (ANOVA), followed by Tukey honest significant difference. In vivo flavoprotein optical imaging and field potential recordings Optical imaging was performed as previously described (27). The mice were anesthetized with urethane (intraperitoneal injection of 1.2 mg/kg urethane, supplemented with 0.3 mg/kg urethane as needed) and mechanically ventilated; the core temperature was maintained. Each mouse was placed in a stereotaxic frame, crus I and II of the cerebellar cortex were exposed, and the dura was removed. An acrylic chamber was constructed around the exposed folia and filled with normal Ringer solution. The mouse and stereotaxic frame were placed on a large stage with precision x and y translation. Flavoprotein-based autofluorescence optical imaging was performed using Nikon epifluorescence optics, with an excitation band-pass filter of 420 to 490 nm, a 500-nm dichroic mirror, and a long-pass filter of >515 nm. The camera was focused just below the surface of the cerebellar cortex, and parallel fibers were activated by a tungsten microelectrode placed just into the molecular layer. The basic imaging paradigm consisted of collecting a time series of 200-ms images before, during, and after parallel fiber (PF) stimulation (a train of 10 pulses at 100 Hz of 200 μA and 100 μs). Having obtained a series of images, the optical response was determined by subtracting the average of the first 20 background images (control images before PF stimulation) from each image in the series acquired to generate a series of “difference” images. These difference images were then divided by the control average on a pixel-by-pixel basis, in which the intensity value of each pixel reflects the change in fluorescence intensity relative to the average of the control frames (ΔF/F). To quantify the response to PF stimulation, a region of interest (ROI), defined by the evoked beam or long-term patches, was visually determined, and the same ROI was used throughout an experiment to quantify any changes in the response. For the beam, the response to the parallel fiber stimulation consisted of an initial increase in fluorescence (light phase) that is tightly coupled to the strength of the stimulation (25). Therefore, the analysis was restricted to the light phase, averaging the five frames centered on the peak amplitude of the light phase to obtain the average ΔF/F within the ROI. The long-latency patches occurred ∼25 to 30 seconds after PF stimulation, an ROI was defined for each patch, and the 25 frames were averaged around the peak to obtain the amplitude (27). Beam and patch intensities during the baseline measurements were compared with those occurring after 50 to 65 minutes of bath-applied drug (or vehicle). The effects were analyzed using a paired Student t test. For the experiments using the metabotropic glutamate receptor (mGluR) 1 antagonist JNJ 16259685 (JNJ; 1 μM) and the estrogen receptor antagonist ICI 182,780 (ICI; 1 μM), fluorescence was measured first for 30 minutes after JNJ treatment and then 30 minutes later in the presence of JNJ and ICI. The effects were analyzed via an ANOVA (within-subject design with repeated measures), followed by a post hoc Bonferroni test. Electrophysiology Field potential recordings Field potential recordings of the responses to PF stimulation provided an electrophysiological assessment of the pre- vs postsynaptic effects of blocking estrogen receptors in the cerebellum of mice. Using established protocols (27), the field potentials in the molecular layer were recorded using glass microelectrodes (2M NaCl, 2 to 5 MΩ), digitized at 25,000 Hz and averaged (responses to 16 single PF stimuli at 1 Hz). The P1/N1 component is a measure of the presynaptic response, and the N2 component is a measure of the postsynaptic response (25, 27, 28). The field potentials were monitored every 3 to 4 minutes for 30 minutes before and for 65 minutes during the drug applications. The field potential amplitudes during the baseline measurements were compared with those after 50 to 65 minutes of bath-applied drugs. The effects of ICI and Fad on the P1/N1 and N2 components were analyzed using ANOVA (within-subject design) followed by a post hoc Bonferroni test. Whole-cell patch clamp Coronal cerebellar slices (400-μm thick) were prepared with a vibroslicer (VT 1200 S; Leica, Germany) guided by the rat brain atlas (29). The slices were incubated in artificial cerebrospinal fluid (ACSF; composition: 124 mM NaCl, 2.5 mM KCl, 1.25 mM NaH2PO4, 1.3 mM MgSO4, 26 mM NaHCO3, 2 mM CaCl2, and 10 mM d-glucose) equilibrated with 95% oxygen and 5% carbon dioxide at 35° ± 0.5°C for ≥1 hour and then maintained at room temperature. During recording sessions, the slices were transferred to a submersion chamber and continuously perfused with 95% oxygen and 5% carbon dioxide–oxygenated ACSF at a rate of 2 mL/min maintained at room temperature. Whole-cell patch recordings were performed as in previous reports (30, 31) on cerebellar cortical Purkinje neurons with borosilicate glass pipettes (3 to 5 MΩ) filled with an internal solution (composition: 140 mM K-methylsulfate, 7 mM KCl, 2 mM MgCl2, 10 mM HEPES, 0.1 mM EGTA, 4 mM Na2-ATP, 0.4 mM GTP-Tris, adjusted to pH 7.25, with 1 M KOH). During the recording sessions, Purkinje neurons were visualized with an Olympus BX51WI microscope. Patch-clamp recordings were acquired with an Axopatch-700B amplifier (Molecular Devices, Sunnyvale, CA). Data capture and analysis was performed using a Digidata-1440A interface and pClamp, version 10.2, software (Molecular Devices). Neurons were held at a membrane potential of −70 mV and characterized by injection of a rectangular voltage pulse (5 mV, 50 ms) to monitor the whole-cell membrane capacitance, series resistance, and membrane resistance. Neurons were excluded from the study if the series resistance was not stable or exceeded 20 MΩ. The slices were bathed in the pan-specific group I mGluR agonist (S)-3,5-dihydroxyphenylglycine (DHPG; 100 μM; Tocris, Minneapolis, MN) to induce an inward current by specific activation of mGluR on the recorded Purkinje neurons. Next, the slices were perfused with ACSF containing 17β-estradiol (1 nM; Sigma-Aldrich) for 20 minutes to observe the effect of estradiol on the mGluR-specific current. Afterward, the actions of estradiol were tested in the presence of the mGluR1-specific antagonist CPCCOEt (125 μM; Tocris) to assess whether the effect of estradiol was dependent on mGluR1. CPCCOEt was used instead of JNJ in these experiments, because the JNJ drug was unavailable to the experimenters at the time. In addition to the pharmacologically induced mGluR current, we determined the effect of estradiol on the slow excitatory postsynaptic currents (sEPSCs) and mGluR excitatory postsynaptic currents (EPSCs) evoked by PF stimulation. A concentric bipolar electrode (CB ARC75; FHC Inc.) was positioned in the molecular layer in the vicinity of the recorded neuron to stimulate the PFs. PFs were activated by applying a train of 10 pulses (20 to 200 μA, 300 μs) at 100 Hz to the electrode through a programmable stimulator (Master-9; A.M.P.I., Jerusalem Israel). The γ-aminobutyric acid A receptor antagonist SR95531 (20 μM) and the glutamate transporter blocker DL-TBOA (50 μM) were applied to inhibit inhibitory synaptic transmission and obtain apparent sEPSCs. The effects of 17β-estradiol (1 nM) on the PF-evoked sEPSCs in the absence and presence of CPCCOEt (125 μM) were evaluated. For whole-cell patch clamp experiments, data were analyzed via a paired Student t test. Estrogen concentration measurements Extraction of estradiol from cerebellar cortex homogenate was adapted from previously reported methods (32). Cerebellar tissue was maintained at −80°C until the day of extraction. Homogenates [one-quarter fraction of tissue in 1 mL of 0.1 M phosphate buffer (PB)] were first liquid extracted with 2 mL of diethyl ether three times in series for maximum yield. After resuspension in 0.1 M PB, the samples were solid phase extracted on C18 columns (Empore) with an elution using 100% MeOH (high-performance liquid chromatography grade). The estradiol concentrations were then measured using a validated enzyme-linked immunosorbent assay (catalog no. 582251; Cayman Chemical, Ann Arbor, MI). The estradiol content measurements from the assay were corrected based on the wet weight (mg) of each cerebellar hemisphere. All samples were detectable above blanks (0.1 M PB with no brain content) with 78.5% extraction efficiency and standard curve R2 at 98%. Results The effect of estrogen depletion on cerebellar functioning was assessed via analysis of locomotor behavior, because the cerebellum is required for normal movement (33) and estrogen increases Purkinje cell activity during locomotion (10, 34). To avoid potential confounders, these behavioral studies solely used male mice, because estrogens are known to affect basal ganglia functioning in females (35), including enhancing sensorimotor performance (36). The behavioral analysis consisted of monitoring treadmill activity via the DigiGait™ System. Both intact and castrated mice were compared. Conspicuously, although the male gonads produce estrogens, they are not the primary hormone produced by the testes. Hence, behavioral changes due to castration alone cannot singly be ascribed to a deficiency in estrogens. Because the brain can also synthesize estrogens directly, as well as can other organs outside the gonads, both intact and castrated mice were implanted with a minipump to systemically deliver either the aromatase inhibitor Fad (0.5 mg/kg/d) or vehicle. Fad administration allowed us to directly attribute alterations in behavior to changes in estradiol availability. Both castration and Fad produced deficits in locomotor performance. Two main behaviors were altered. When examining the ratio in which the rear paws were in a stance position vs swinging forward (Fig. 1A), main effects were found for both castration (F = 16.53; P < 0.01, n = 4 to 10 per group) and Fad (F = 7.08; P < 0.05). Similar results for castration (F = 13.08; P < 0.01) and Fad (F = 5.55, P < 0.05) were found when examining the average time in which both rear paws were on the ground (Fig. 1B). Of particular interest, deficits in these same behaviors were observed in a mouse model of cerebellar ataxia (37). Figure 1. View largeDownload slide Diminution of estrogen signaling adversely affects cerebellum-dependent motor behavior. (A and B) DigiGait™ analysis of locomotor activity in both intact and castrated (CAST) male mice after chronic administration of Fad or vehicle. Both castration and Fad decreased locomotor performance when quantifying the ratio of stance to swing of the (A) rear paw and (B) shared rear paw stance time (*P < 0.05; **P < 0.01). Data presented as mean ± standard error of the mean. Figure 1. View largeDownload slide Diminution of estrogen signaling adversely affects cerebellum-dependent motor behavior. (A and B) DigiGait™ analysis of locomotor activity in both intact and castrated (CAST) male mice after chronic administration of Fad or vehicle. Both castration and Fad decreased locomotor performance when quantifying the ratio of stance to swing of the (A) rear paw and (B) shared rear paw stance time (*P < 0.05; **P < 0.01). Data presented as mean ± standard error of the mean. In attempt to uncover the mechanism by which estradiol affects cerebellar functioning, we turned to activity-dependent optical imaging. Using this technique, we were able to access, in vivo, alterations in cerebellar synaptic signaling. Specifically, changes in the endogenous fluorescence of mitochondrial flavoproteins were used to monitor PF–Purkinje cell synaptic transmission in the anesthetized mouse (Fig. 2A). The PFs are the bifurcated axons of the granule cells and create 100,000 to 200,000 glutamatergic synapses on each postsynaptic Purkinje cell (28). Furthermore, estradiol supports glutamatergic neurotransmission in the cerebellum (7–10). PFs were stimulated with a train of 10 pulses (200 μA, 100 μs) at 100 Hz using a tungsten microelectrode. This stimulation protocol produces two distinct patterns of fluorescent activity, both dependent on glutamatergic neurotransmission. The initial beam-like increase in flavoprotein fluorescence in response to PF stimulation is due to the postsynaptic activation of Purkinje cells and is principally dependent on AMPA receptors (AMPARs), with a smaller contribution from the group I mGluR, mGluR1a (25, 38). On the decay of the beam fluorescent signal, a second fluorescent signal arises, specifically, patches of increased fluorescent activity, driven principally by activation of mGluR1a (27). Figure 2. View largeDownload slide Estrogen receptor activity supports glutamatergic neurotransmission between cerebellar granule cells and Purkinje neurons. (A) Illustration of the experimental setup. (Left) The electrode activates granule cell PFs, initiating glutamatergic neurotransmission across multiple Purkinje neurons. (Middle) PF stimulation evokes an initial beam of fluorescent activity that is primarily dependent on AMPAR activation with a smaller group I mGluR-dependent component, followed several seconds later by (Right) several patches solely reliant on group I mGluR activation. (B) Paired bright-field and ΔF/F images of an exposed cerebellum in which PF activation was initiated with a stimulating electrode (lower right-hand corner of the bright-field image). Shown are the ΔF/F images of the evoked beam and patch responses before and after administration of the estrogen receptor antagonist ICI (1 μM). (C and D) Within 30 minutes of bath application directly to the cerebellum, ICI produced a decrease in both beam and patch responses (**P < 0.01). (E) Paired bright-field and ΔF/F images of a cerebellum exposed to the GPR30 agonist G1 (1 μM). (F and G) G1 had no effect on either the beam or the patch responses. Data presented as mean ± standard error of the mean. Figure 2. View largeDownload slide Estrogen receptor activity supports glutamatergic neurotransmission between cerebellar granule cells and Purkinje neurons. (A) Illustration of the experimental setup. (Left) The electrode activates granule cell PFs, initiating glutamatergic neurotransmission across multiple Purkinje neurons. (Middle) PF stimulation evokes an initial beam of fluorescent activity that is primarily dependent on AMPAR activation with a smaller group I mGluR-dependent component, followed several seconds later by (Right) several patches solely reliant on group I mGluR activation. (B) Paired bright-field and ΔF/F images of an exposed cerebellum in which PF activation was initiated with a stimulating electrode (lower right-hand corner of the bright-field image). Shown are the ΔF/F images of the evoked beam and patch responses before and after administration of the estrogen receptor antagonist ICI (1 μM). (C and D) Within 30 minutes of bath application directly to the cerebellum, ICI produced a decrease in both beam and patch responses (**P < 0.01). (E) Paired bright-field and ΔF/F images of a cerebellum exposed to the GPR30 agonist G1 (1 μM). (F and G) G1 had no effect on either the beam or the patch responses. Data presented as mean ± standard error of the mean. To determine the role of estrogen receptor signaling, measurements of beam and patch signal intensity were compared before and after administration of the estrogen receptor antagonist ICI (1 μM) to the ACSF-filled optical chamber (Fig. 2B). In cerebella from gonadally intact male mice, ICI produced a statistically significant decrease in both the beam (T = 4.84; P < 0.01; n = 5; Fig. 2C) and patch (T = 12.28; P < 0.01; Fig. 2D) fluorescent responses. In contrast, administration of vehicle to the optical chamber had no effect on either measure (n = 4; data not shown). To verify this was not a male-specific effect, the same measurements were obtained in the cerebella of ovariectomized female animals (n = 3), where ICI produced a statistically significant decrease in beam (control, 1.89% ± 0.05% vs ICI, 1.38% ± 0.03% ΔF/F; T = 5.54; P < 0.01) and patch (control, 1.29% ± 0.15% vs ICI, 0.49% ± 0.16% ΔF/F; T = 2.19; P < 0.05) fluorescence. In addition to antagonism of classic estrogen receptors, ICI has also been demonstrated to activate G protein–coupled receptor homolog (GPR30) (39). Thus, to ascertain whether the effect of ICI could be attributed to this alternative receptor, we applied the GPR30 agonist G1 (1 μM) directly to the optical chamber in five male mice (Fig. 2E). G1 was found to have no effect on either the beam (T = 1.43; P = NS; Fig. 2F) or patch (T = 1.48; P = NS; Fig. 2G) response. Decreased activity in both the beam and the patch response by ICI indicated either an estrogen-mediated effect on mGluR signaling alone or an action on both mGluRs and AMPARs. To further explore how estrogen receptor activation is critical for the maintenance of synaptic transmission between PFs and Purkinje cells, the effect of ICI in the presence of the mGluR1a antagonist JNJ (1 μM) was examined in male mice. As expected, JNJ by itself produced a small, but statistically significant, decrease in beam amplitude (F = 9.26; P < 0.01; n = 5; Fig. 3A and 3B), with concurrent elimination of the patch response (F = 15.31; P < 0.01; Fig. 3C). However, ICI produced no additional effect in the presence of JNJ. These data indicate an effect of estrogen dependent on mGluR1a without affecting AMPAR signaling. Although it is well established that in the female rodent nervous system, estradiol activation of membrane-localized estrogen receptors leads to stimulation of group I mGluRs (40), to the best of our knowledge, these are the first experimental data from male rodents to indicate a brain region in the male that responds similarly. These data are also consistent with previous observations in the male quail brain (41). Figure 3. View largeDownload slide Estrogen receptor regulation of PF–Purkinje cell neurotransmission is dependent on group I mGluRs. (A) Paired bright-field and ΔF/F images of an exposed cerebellum in which PF stimulation resulted in beam and patch responses. Baseline fluorescent responses were compared with the responses when the cerebellum was treated first with the group I mGluR antagonist JNJ (1 μM), followed by the combination of JNJ and ICI. (B and C) The effect of estrogen receptor antagonism on the beam and patch responses to PF stimulation was eliminated after inhibition of group I mGluRs. The differences in the results of both treatment groups compared with baseline were statistically significant (**P < 0.01) but were not substantially different from each other. Data presented as mean ± standard error of the mean. Figure 3. View largeDownload slide Estrogen receptor regulation of PF–Purkinje cell neurotransmission is dependent on group I mGluRs. (A) Paired bright-field and ΔF/F images of an exposed cerebellum in which PF stimulation resulted in beam and patch responses. Baseline fluorescent responses were compared with the responses when the cerebellum was treated first with the group I mGluR antagonist JNJ (1 μM), followed by the combination of JNJ and ICI. (B and C) The effect of estrogen receptor antagonism on the beam and patch responses to PF stimulation was eliminated after inhibition of group I mGluRs. The differences in the results of both treatment groups compared with baseline were statistically significant (**P < 0.01) but were not substantially different from each other. Data presented as mean ± standard error of the mean. The reduction of mGluR1a activation after ICI treatment could be due to alterations in pre- and/or postsynaptic activity of the PF synapse. To distinguish between these possibilities, field potential recordings of the response to PF stimulation were compared before and after the ICI addition to the bath (Fig. 4). Analysis of the field potentials (Fig. 4B) indicated a statistically significant effect of ICI (F = 12.35; P < 0.05; n = 4), but only the postsynaptic N2 component significantly decreased (P < 0.01, Bonferroni post hoc) without a substantial effect on the presynaptic P1/N1 component. These experiments were repeated using female mice (n = 4; Fig. 4C). ICI had a similar effect in females (F = 20.24; P < 0.05) as in males, with a reduction only in the N2 (P < 0.01) and not in the P1/N1 component. Figure 4. View largeDownload slide Estrogen receptor signaling is mediated by postsynaptic activity. (A) Field recordings of the responses to parallel fiber stimulation (1 Hz with 100 μA and100 μs pulses; average of 16 trials) before and after application of ICI. (B and C) ICI decreased the postsynaptic N2 component of the electrical recording (**P < 0.01) without affecting the presynaptic P1/N1 components in both (B) male and (C) female mice. Data presented as mean ± standard error of the mean. Figure 4. View largeDownload slide Estrogen receptor signaling is mediated by postsynaptic activity. (A) Field recordings of the responses to parallel fiber stimulation (1 Hz with 100 μA and100 μs pulses; average of 16 trials) before and after application of ICI. (B and C) ICI decreased the postsynaptic N2 component of the electrical recording (**P < 0.01) without affecting the presynaptic P1/N1 components in both (B) male and (C) female mice. Data presented as mean ± standard error of the mean. Previous studies of females demonstrate estrogen receptor activity can lead to either mGluR1a or mGluR5 signaling, dependent on the brain site (42–46). Adult Purkinje neurons typically express only mGluR1a (47, 48), although under some conditions mGluR5 expression is also observed (49). Hence, the next experiment was designed to corroborate previous work by testing whether estrogen signaling in Purkinje neurons was dependent on mGluR1a. In addition, the experimental method was to apply estradiol to determine whether it would enhance glutamatergic signaling in rats, similar in scope to the work previously described by Smith et al. (7–10). Whole-cell recordings of Purkinje neurons from cerebellar slices in both male (n = 3) and female (n = 2) animals examined the acute effect of estradiol (1 nM) on DHPG-induced (100 μM) inward currents (Fig. 5). The inward current produced by the pan-specific group I mGluR agonist was potentiated by estradiol (T = 9.35; P < 0.01, n = 5 recordings, one from each animal). Also, consistent with our hypotheses, application of the mGluR1-specific antagonist CPCCOEt (125 μM) both blocked ∼90% of the total DHPG-induced current and eliminated any effect by the hormone, indicating that in Purkinje cells, estradiol affects cerebellar neurotransmission through modulation of mGluR1a. The residual current insensitive to CPCCOEt might be mGluR5 mediated, because the recordings were taken from animals at an age in which the cerebellum does express mGluR5 (50). Figure 5. View largeDownload slide Estradiol enhanced mGluR1a activity in cerebellar Purkinje neurons. (A) A whole-cell recording from a cerebellar Purkinje neuron. Estradiol (1 nM) potentiated the inward current induced by the group I mGluR1 agonist DHPG (100 μM). Isolation of mGluR5 after bath application of the mGluR1 antagonist CPCCOEt (125 μM) eliminated the effect of estradiol, demonstrating estradiol-mediated enhancement of mGluR1a. (B) Group data demonstrating estradiol-mediated enhancement of DHPG-induced current (**P < 0.01) is dependent on mGluR1a. (C) Estradiol potentiation of sEPSCs is dependent on mGluR1a. A whole-cell recording of sEPSCs in a Purkinje neuron after stimulation of the molecular layer. Estradiol (1 nM) enhancement of the sEPSC was eliminated after administration of the mGluR1 antagonist CPCCOEt. (D) Group data demonstrating estradiol-mediated enhancement of sEPSCs (**P < 0.01) is dependent on mGluR1a. Data presented as mean ± standard error of the mean. Figure 5. View largeDownload slide Estradiol enhanced mGluR1a activity in cerebellar Purkinje neurons. (A) A whole-cell recording from a cerebellar Purkinje neuron. Estradiol (1 nM) potentiated the inward current induced by the group I mGluR1 agonist DHPG (100 μM). Isolation of mGluR5 after bath application of the mGluR1 antagonist CPCCOEt (125 μM) eliminated the effect of estradiol, demonstrating estradiol-mediated enhancement of mGluR1a. (B) Group data demonstrating estradiol-mediated enhancement of DHPG-induced current (**P < 0.01) is dependent on mGluR1a. (C) Estradiol potentiation of sEPSCs is dependent on mGluR1a. A whole-cell recording of sEPSCs in a Purkinje neuron after stimulation of the molecular layer. Estradiol (1 nM) enhancement of the sEPSC was eliminated after administration of the mGluR1 antagonist CPCCOEt. (D) Group data demonstrating estradiol-mediated enhancement of sEPSCs (**P < 0.01) is dependent on mGluR1a. Data presented as mean ± standard error of the mean. To verify estradiol potentiation of mGluR1a signaling, a second whole-cell patch clamp experiment was performed to determine the effect of estradiol on PF-evoked EPSCs in Purkinje cells in the absence or presence of the mGluR1 antagonist CPCCOEt (Fig. 6). To that end, we exploited the fact that group I mGluR signaling can enhance electrically evoked EPSCs (51). Consistent with estradiol specifically affecting mGluR1a signaling, estradiol, in both male and female neurons, enhanced EPSCs under control conditions (T = 6.11; P < 0.01) but had no effect in the presence of CPCCOEt (n = 6 recordings; n = 3 separate animals for each sex). Figure 6. View largeDownload slide Local estrogen synthesis facilitates cerebellar PF–Purkinje cell neurotransmission. (A) Paired bright-field and ΔF/F images of the beam and patch responses to PF simulation in a castrated (CAST) mouse. Optical responses were obtained before and after administration of the aromatase inhibitor Fad (1 μM). (B and C) Within 45 minutes of bath application, Fad produced a decrease in both beam and patch (**P < 0.01) signals. (D) Paired bright-field and ΔF/F images of the beam and patch responses to PF simulation in an ovariectomized (OVX) mouse. (E and F) Similar to castrated mice, Fad produced a decrease in both beam (*P < 0.05) and patch (**P < 0.01) responses in ovariectomized mice. Data presented as mean ± standard error of the mean. Figure 6. View largeDownload slide Local estrogen synthesis facilitates cerebellar PF–Purkinje cell neurotransmission. (A) Paired bright-field and ΔF/F images of the beam and patch responses to PF simulation in a castrated (CAST) mouse. Optical responses were obtained before and after administration of the aromatase inhibitor Fad (1 μM). (B and C) Within 45 minutes of bath application, Fad produced a decrease in both beam and patch (**P < 0.01) signals. (D) Paired bright-field and ΔF/F images of the beam and patch responses to PF simulation in an ovariectomized (OVX) mouse. (E and F) Similar to castrated mice, Fad produced a decrease in both beam (*P < 0.05) and patch (**P < 0.01) responses in ovariectomized mice. Data presented as mean ± standard error of the mean. With estrogen receptor activation of mGluR1a signaling required for maximal PF–Purkinje cell neurotransmission, we sought to revisit whether local estrogen synthesis contributes to this process. Cerebellar fractions from intact male mice contained 5.92 ± 3.29 pg/mg estradiol, which were not different from those of castrated mice (8.81 ± 3.19 pg/mg; n = 8/group; T = 0.63; P = NS; assay blanks were 0.04 pg), indicating sources of estrogen were available to the cerebellum independent of the gonads. Parallel to the interaction observed between castration and Fad treatment on locomotor behavior, we next sought to directly test whether cerebellar neuroestrogen synthesis contributed to PF–Purkinje cell neurotransmission. Thus, beam and patch fluorescence in castrated animals were examined after direct administration of Fad (1 μM) to the cerebellar cortex (Fig. 6A–6C). Fad produced a reduction in both beam (T = 7.21; P < 0.01; n = 5) and patch (T = 4.59; P < .01) responses. However, the effects of Fad were not dependent on castration, because in intact male animals (n = 4), Fad also produced a statistically significant reduction to the beam (control, 0.68% ± 0.09% vs Fad, 0.25% ± 0.05% ΔF/F; T = 9.41; P < 0.01) and patch (control, 0.40% ± 0.09% vs Fad, 0.05% ± 0.03% ΔF/F, T = 5.51; P < 0.01) responses. Finally, similar effects were also observed in the cerebella of ovariectomized animals (Fig. 6D–6F), with Fad producing a reduction in both beam (T = 3.85; P < 0.05) and patch (T = 13.12; P < 0.01) responses (n = 4). Discussion The present data support the conclusion that estrogen signaling is critical for both PF–Purkinje cell synaptic transmission and cerebellum-dependent motoric behaviors. Not only does estrogen regulation of Purkinje neuron mGluR1a signaling appear to be a component of normal cerebellar function, but it also displays no overt sex differences, with many similar effects observed in both males and females. Moreover, the cerebellar cortex appears to be a source of estradiol, furthering the notion that this hormone can act locally as a neuromodulator. As such, estradiol regulation of mGluR1a signaling in support of the PF–Purkinje cell synapse provides an underlying mechanism to understand a decades-long uncertainty regarding estrogen function within the adult cerebellum. The concept that estrogen supports basal cerebellar neurotransmission might seem heretical. However, accumulating evidence has been consistent with this hypothesis. Estrogen enhancement of cerebellar glutamatergic neurotransmission, the expression of aromatase within the cerebellum, and the dysfunction of behaviors attributable to cerebellar performance after administration of aromatase inhibitors are three distinct lines of evidence that support this hypothesis (52). Uncovering estrogen regulation of mGluR1a signaling as a mechanism by which this steroid hormone acts within the cerebellum integrates these previous studies. It should be noted, however, that although our data are consistent with this hypothesis, alternative explanations exist. Owing to the nature of behavioral studies, we cannot unequivocally rule out effects of Fad in other brain regions or the periphery. Second, the level of aromatase inhibition achieved by central administration of fadrozole is unknown. The observed effects could not only be due to a decrease in estradiol but also to an increase in testosterone. Finally, although estrogen receptor coupling to mGluR1a has been demonstrated in various systems (42, 46, 53, 54), estrogen receptor activation could facilitate mGluR1a function through a different pathway. From various experimental systems, we know that estradiol synthesis occurs within the nervous system, can exert effects at the cell membrane in response to a stimulus, and exhibits enzymatic inactivation to terminate signaling. Based on these benchmarks, it has been hypothesized that estradiol can act as a neurotransmitter (55, 56). Our findings regarding the diminution of locomotor behavior and PF–Purkinje cell synaptic transmission after depletion of cerebellar estrogens without directly manipulating the glutamate concentrations within this brain region further support this model. The work by Smith et al. (8, 10) first demonstrated that estrogens enhance glutamatergic neurotransmission in the cerebellum, with a corresponding increase in Purkinje cell activity during locomotor behavior. However, this phenomenon might not be unique to the cerebellum, because estradiol regulation of glutamatergic neurotransmission and connectivity has been reported in several other brain regions (57–60). The decreased locomotor performance of male mice after Fad administration is consistent with data from human studies in which some women administered aromatase inhibitors have exhibited ataxia (61). Importantly, the cerebellum is involved in more than just motor-related behaviors, including language, executive function, attention, working memory, pain, emotion, and addiction (62–64). Estrogen signaling in the cerebellum might influence memory (11, 12), cognition (13–16), and mood (65). It is possible that these and other influences underlie the low adherence rate of patients taking tamoxifen therapy, although a lack of compliance results in a striking increase in the mortality rate (66–70). Through pharmacological manipulations that circumvent the steroid hormone receptor, it might be possible to gain novel therapeutic strategies to inhibit estrogen-responsive cancers with concurrent maintenance of nervous system function. Local estrogen regulation of mGluR signaling in the Purkinje neuron is most likely just one of several actions of estradiol in the cerebellum. Endogenous estradiol affects Purkinje cell dendritic growth, spine density, synaptogenesis, and excitability (4–6) and enhances long-term potentiation and vestibulo-ocular reflexes (71). Cerebellar estrogen signaling also provides neuroprotection from a variety of insults (72, 73). Its supplementation preserves the cerebellar gray matter as a function of aging (74, 75) and might improve locomotor and cognitive function in both disease and aging paradigms (76–78). Future work will need to gain a greater appreciation for the role estradiol plays in maintaining and modulating cerebellar function during development, in the adult, and in disease states. In conclusion, estradiol promotion of the PF–Purkinje cell synapse is an aspect of cerebellar physiology. Furthermore, the cerebellum appears capable of synthesizing estrogens to ensure proper neurotransmission, and deficits in estrogen production appear to adversely affect cerebellar function. Abbreviations: Abbreviations: ACSF artificial cerebrospinal fluid AMPAR AMPA receptor ANOVA analysis of variance DHPG (S)-3,5-dihydroxyphenylglycine EPSC excitatory postsynaptic current Fad fadrozole GPR30 G protein–coupled receptor homolog ICI ICI 182,780 JNJ JNJ 16259685 PF parallel fiber ROI region of interest mGluR metabotropic glutamate receptor mGluR1a metabotropic glutamate receptor type 1a sEPSC slow excitatory postsynaptic current ΔF/F change in fluorescence intensity relative to the average of the control frames Acknowledgments The authors thank Dr. Robert Meisel for his comments and suggestions pertaining to the present work. Financial Support: This work was supported by the National Institute of Neurologic Disorders and Stroke (Grant NS077661 to P.G.M. and T.J.E.; Grants NS062158 and NS18338 to T.J.E.; and Grants NS066179 and NS082179 to L.R.-H.); the National Institute on Drug Abuse (Grants DA035008 and DA041808 to P.G.M.); the National Natural Science Foundation of China (Grants 31330033, 91332124, and 31461163001 to J.-J.W.; and Grants 81671107 and 31471112 to J.-N.Z.); the Specialized Research Fund for the Doctoral Program of Higher Education and Research Grants Council Earmarked Research Grants (Grant 20130091140003 to J.-J.W.); and the Jiangsu Natural Science Foundation (Grant BK2011014 to J.-J.W.). Disclosure Summary: The authors have nothing to disclose. References 1. McEwen BS, Pfaff DW. Factors influencing sex hormone uptake by rat brain regions. I. Effects of neonatal treatment, hypophysectomy, and competing steroid on estradiol uptake. Brain Res . 1970; 21( 1): 1– 16. Google Scholar CrossRef Search ADS PubMed  2. Eleftheriou BE, Desjardins C, Pattison ML. Effects of ovariectomy and ovarian hormones on RNA base ratios in specific rabbit brain areas. J Endocrinol . 1970; 46( 3): 331– 340. Google Scholar CrossRef Search ADS PubMed  3. Greengrass PM, Tonge SR. Suggestions on the pharmacological actions of ethinyloestradiol and progesterone on the control of monoamine metabolism in three regions from the brains of gonadectomized male and female mice and the possible clinical significance. Arch Int Pharmacodyn Ther . 1974; 211( 2): 291– 304. Google Scholar PubMed  4. Sakamoto H, Mezaki Y, Shikimi H, Ukena K, Tsutsui K. Dendritic growth and spine formation in response to estrogen in the developing Purkinje cell. Endocrinology . 2003; 144( 10): 4466– 4477. Google Scholar CrossRef Search ADS PubMed  5. Sasahara K, Shikimi H, Haraguchi S, Sakamoto H, Honda S, Harada N, Tsutsui K. Mode of action and functional significance of estrogen-inducing dendritic growth, spinogenesis, and synaptogenesis in the developing Purkinje cell. J Neurosci . 2007; 27( 28): 7408– 7417. Google Scholar CrossRef Search ADS PubMed  6. Dean SL, Wright CL, Hoffman JF, Wang M, Alger BE, McCarthy MM. Prostaglandin E2 stimulates estradiol synthesis in the cerebellum postnatally with associated effects on Purkinje neuron dendritic arbor and electrophysiological properties. Endocrinology . 2012; 153( 11): 5415– 5427. Google Scholar CrossRef Search ADS PubMed  7. Smith SS. Estrogen administration increases neuronal responses to excitatory amino acids as a long-term effect. Brain Res . 1989; 503( 2): 354– 357. Google Scholar CrossRef Search ADS PubMed  8. Smith SS, Waterhouse BD, Woodward DJ. Locally applied estrogens potentiate glutamate-evoked excitation of cerebellar Purkinje cells. Brain Res . 1988; 475( 2): 272– 282. Google Scholar CrossRef Search ADS PubMed  9. Smith SS, Waterhouse BD, Woodward DJ. Sex steroid effects on extrahypothalamic CNS. I. Estrogen augments neuronal responsiveness to iontophoretically applied glutamate in the cerebellum. Brain Res . 1987; 422( 1): 40– 51. Google Scholar CrossRef Search ADS PubMed  10. Smith SS, Woodward DJ, Chapin JK. Sex steroids modulate motor-correlated increases in cerebellar discharge. Brain Res . 1989; 476( 2): 307– 316. Google Scholar CrossRef Search ADS PubMed  11. Phillips SM, Sherwin BB. Variations in memory function and sex steroid hormones across the menstrual cycle. Psychoneuroendocrinology . 1992; 17( 5): 497– 506. Google Scholar CrossRef Search ADS PubMed  12. Phillips SM, Sherwin BB. Effects of estrogen on memory function in surgically menopausal women. Psychoneuroendocrinology . 1992; 17( 5): 485– 495. Google Scholar CrossRef Search ADS PubMed  13. Rocca WA, Bower JH, Maraganore DM, Ahlskog JE, Grossardt BR, de Andrade M, Melton LJ III. Increased risk of cognitive impairment or dementia in women who underwent oophorectomy before menopause. Neurology . 2007; 69( 11): 1074– 1083. Google Scholar CrossRef Search ADS PubMed  14. Schilder CM, Seynaeve C, Beex LV, Boogerd W, Linn SC, Gundy CM, Huizenga HM, Nortier JW, van de Velde CJ, van Dam FS, Schagen SB. Effects of tamoxifen and exemestane on cognitive functioning of postmenopausal patients with breast cancer: results from the neuropsychological side study of the tamoxifen and exemestane adjuvant multinational trial. J Clin Oncol . 2010; 28( 8): 1294– 1300. Google Scholar CrossRef Search ADS PubMed  15. Schilder CM, Seynaeve C, Linn SC, Boogerd W, Beex LV, Gundy CM, Nortier JW, van de Velde CJ, van Dam FS, Schagen SB. Cognitive functioning of postmenopausal breast cancer patients before adjuvant systemic therapy, and its association with medical and psychological factors. Crit Rev Oncol Hematol . 2010; 76( 2): 133– 141. Google Scholar CrossRef Search ADS PubMed  16. Schilder CM, Seynaeve C, Linn SC, Boogerd W, Gundy CM, Beex LV, van Dam FS, Schagen SB. The impact of different definitions and reference groups on the prevalence of cognitive impairment: a study in postmenopausal breast cancer patients before the start of adjuvant systemic therapy. Psychooncology . 2010; 19( 4): 415– 422. Google Scholar CrossRef Search ADS PubMed  17. Rewal M, Wen Y, Simpkins JW, Jung ME. Ethanol withdrawal reduces cerebellar parvalbumin expression in a manner reversed by estrogens. Neurosci Lett . 2005; 377( 1): 44– 48. Google Scholar CrossRef Search ADS PubMed  18. Mirzatoni A, Spence RD, Naranjo KC, Saldanha CJ, Schlinger BA. Injury-induced regulation of steroidogenic gene expression in the cerebellum. J Neurotrauma . 2010; 27( 10): 1875– 1882. Google Scholar CrossRef Search ADS PubMed  19. Zorrilla Zubilete MA, Guelman LR, Maur DG, Caceres LG, Rios H, Zieher LM, Genaro AM. Partial neuroprotection by 17-β-estradiol in neonatal γ-irradiated rat cerebellum. Neurochem Int . 2011; 58( 3): 273– 280. Google Scholar CrossRef Search ADS PubMed  20. Firozan B, Goudarzi I, Elahdadi Salmani M, Lashkarbolouki T, Rezaei A, Abrari K. Estradiol increases expression of the brain-derived neurotrophic factor after acute administration of ethanol in the neonatal rat cerebellum. Eur J Pharmacol . 2014; 732: 1– 11. Google Scholar CrossRef Search ADS PubMed  21. Mermelstein PG, Micevych PE. Nervous system physiology regulated by membrane estrogen receptors. Rev Neurosci . 2008; 19( 6): 413– 424. Google Scholar CrossRef Search ADS PubMed  22. Kelly MJ, Rønnekleiv OK. Membrane-initiated actions of estradiol that regulate reproduction, energy balance and body temperature. Front Neuroendocrinol . 2012; 33( 4): 376– 387. Google Scholar CrossRef Search ADS PubMed  23. Biegon A, Kim SW, Alexoff DL, Jayne M, Carter P, Hubbard B, King P, Logan J, Muench L, Pareto D, Schlyer D, Shea C, Telang F, Wang GJ, Xu Y, Fowler JS. Unique distribution of aromatase in the human brain: in vivo studies with PET and [N-methyl-11C]vorozole. Synapse . 2010; 64( 11): 801– 807. Google Scholar CrossRef Search ADS PubMed  24. Azcoitia I, Yague JG, Garcia-Segura LM. Estradiol synthesis within the human brain. Neuroscience . 2011; 191: 139– 147. Google Scholar CrossRef Search ADS PubMed  25. Reinert KC, Dunbar RL, Gao W, Chen G, Ebner TJ. Flavoprotein autofluorescence imaging of neuronal activation in the cerebellar cortex in vivo. J Neurophysiol . 2004; 92( 1): 199– 211. Google Scholar CrossRef Search ADS PubMed  26. Reinert KC, Gao W, Chen G, Wang X, Peng YP, Ebner TJ. Cellular and metabolic origins of flavoprotein autofluorescence in the cerebellar cortex in vivo. Cerebellum . 2011; 10( 3): 585– 599. Google Scholar CrossRef Search ADS PubMed  27. Wang X, Chen G, Gao W, Ebner TJ. Parasagittally aligned, mGluR1-dependent patches are evoked at long latencies by parallel fiber stimulation in the mouse cerebellar cortex in vivo. J Neurophysiol . 2011; 105( 4): 1732– 1746. Google Scholar CrossRef Search ADS PubMed  28. Eccles JC. The Cerebellum as a Neuronal Machine. Berlin: Springer-Verlag; 1967. 29. Paxinos G, Watson C. The Rat Brain in Stereotaxic Coordinates . 6th ed. New York: Academic Press. 30. Zhang J, Li B, Yu L, He YC, Li HZ, Zhu JN, Wang JJ. A role for orexin in central vestibular motor control. Neuron . 2011; 69( 4): 793– 804. Google Scholar CrossRef Search ADS PubMed  31. Zhang XY, Yu L, Zhuang QX, Peng SY, Zhu JN, Wang JJ. Postsynaptic mechanisms underlying the excitatory action of histamine on medial vestibular nucleus neurons in rats. Br J Pharmacol . 2013; 170( 1): 156– 169. Google Scholar CrossRef Search ADS PubMed  32. Chao A, Schlinger BA, Remage-Healey L. Combined liquid and solid-phase extraction improves quantification of brain estrogen content. Front Neuroanat . 2011; 5: 57. Google Scholar CrossRef Search ADS PubMed  33. Glickstein M, Strata P, Voogd J. Cerebellum: history. Neuroscience . 2009; 162( 3): 549– 559. Google Scholar CrossRef Search ADS PubMed  34. Smith SS. Sensorimotor-correlated discharge recorded from ensembles of cerebellar Purkinje cells varies across the estrous cycle of the rat. J Neurophysiol . 1995; 74( 3): 1095– 1108. Google Scholar CrossRef Search ADS PubMed  35. Becker JB. Gender differences in dopaminergic function in striatum and nucleus accumbens. Pharmacol Biochem Behav . 1999; 64( 4): 803– 812. Google Scholar CrossRef Search ADS PubMed  36. Becker JB, Snyder PJ, Miller MM, Westgate SA, Jenuwine MJ. The influence of estrous cycle and intrastriatal estradiol on sensorimotor performance in the female rat. Pharmacol Biochem Behav . 1987; 27( 1): 53– 59. Google Scholar CrossRef Search ADS PubMed  37. Du X, Wang J, Zhu H, Rinaldo L, Lamar KM, Palmenberg AC, Hansel C, Gomez CM. Second cistron in CACNA1A gene encodes a transcription factor mediating cerebellar development and SCA6. Cell . 2013; 154( 1): 118– 133. Google Scholar CrossRef Search ADS PubMed  38. Wang X, Chen G, Gao W, Ebner T. Long-term potentiation of the responses to parallel fiber stimulation in mouse cerebellar cortex in vivo. Neuroscience . 2009; 162( 3): 713– 722. Google Scholar CrossRef Search ADS PubMed  39. Filardo EJ, Quinn JA, Bland KI, Frackelton AR Jr. Estrogen-induced activation of Erk-1 and Erk-2 requires the G protein-coupled receptor homolog, GPR30, and occurs via trans-activation of the epidermal growth factor receptor through release of HB-EGF. Mol Endocrinol . 2000; 14( 10): 1649– 1660. Google Scholar CrossRef Search ADS PubMed  40. Meitzen J, Mermelstein PG. Estrogen receptors stimulate brain region specific metabotropic glutamate receptors to rapidly initiate signal transduction pathways. J Chem Neuroanat . 2011; 42( 4): 236– 241. Google Scholar CrossRef Search ADS PubMed  41. Seredynski AL, Balthazart J, Ball GF, Cornil CA. Estrogen receptor β activation rapidly modulates male sexual motivation through the transactivation of metabotropic glutamate receptor 1a. J Neurosci . 2015; 35( 38): 13110– 13123. Google Scholar CrossRef Search ADS PubMed  42. Boulware MI, Weick JP, Becklund BR, Kuo SP, Groth RD, Mermelstein PG. Estradiol activates group I and II metabotropic glutamate receptor signaling, leading to opposing influences on cAMP response element-binding protein. J Neurosci . 2005; 25( 20): 5066– 5078. Google Scholar CrossRef Search ADS PubMed  43. Grove-Strawser D, Boulware MI, Mermelstein PG. Membrane estrogen receptors activate the metabotropic glutamate receptors mGluR5 and mGluR3 to bidirectionally regulate CREB phosphorylation in female rat striatal neurons. Neuroscience . 2010; 170( 4): 1045– 1055. Google Scholar CrossRef Search ADS PubMed  44. Christensen A, Dewing P, Micevych P. Membrane-initiated estradiol signaling induces spinogenesis required for female sexual receptivity. J Neurosci . 2011; 31( 48): 17583– 17589. Google Scholar CrossRef Search ADS PubMed  45. Chaban V, Li J, McDonald JS, Rapkin A, Micevych P. Estradiol attenuates the adenosine triphosphate-induced increase of intracellular calcium through group II metabotropic glutamate receptors in rat dorsal root ganglion neurons. J Neurosci Res . 2011; 89( 11): 1707– 1710. Google Scholar CrossRef Search ADS PubMed  46. Huang GZ, Woolley CS. Estradiol acutely suppresses inhibition in the hippocampus through a sex-specific endocannabinoid and mGluR-dependent mechanism. Neuron . 2012; 74( 5): 801– 808. Google Scholar CrossRef Search ADS PubMed  47. Masu M, Tanabe Y, Tsuchida K, Shigemoto R, Nakanishi S. Sequence and expression of a metabotropic glutamate receptor. Nature . 1991; 349( 6312): 760– 765. Google Scholar CrossRef Search ADS PubMed  48. Baude A, Nusser Z, Roberts JD, Mulvihill E, McIlhinney RA, Somogyi P. The metabotropic glutamate receptor (mGluR1 alpha) is concentrated at perisynaptic membrane of neuronal subpopulations as detected by immunogold reaction. Neuron . 1993; 11( 4): 771– 787. Google Scholar CrossRef Search ADS PubMed  49. Fazio F, Notartomaso S, Aronica E, Storto M, Battaglia G, Vieira E, Gatti S, Bruno V, Biagioni F, Gradini R, Nicoletti F, Di Marco R. Switch in the expression of mGlu1 and mGlu5 metabotropic glutamate receptors in the cerebellum of mice developing experimental autoimmune encephalomyelitis and in autoptic cerebellar samples from patients with multiple sclerosis. Neuropharmacology . 2008; 55( 4): 491– 499. Google Scholar CrossRef Search ADS PubMed  50. Casabona G, Knöpfel T, Kuhn R, Gasparini F, Baumann P, Sortino MA, Copani A, Nicoletti F. Expression and coupling to polyphosphoinositide hydrolysis of group I metabotropic glutamate receptors in early postnatal and adult rat brain. Eur J Neurosci . 1997; 9( 1): 12– 17. Google Scholar CrossRef Search ADS PubMed  51. Dzubay JA, Otis TS. Climbing fiber activation of metabotropic glutamate receptors on cerebellar Purkinje neurons. Neuron . 2002; 36( 6): 1159– 1167. Google Scholar CrossRef Search ADS PubMed  52. Hedges VL, Ebner TJ, Meisel RL, Mermelstein PG. The cerebellum as a target for estrogen action. Front Neuroendocrinol . 2012; 33( 4): 403– 411. Google Scholar CrossRef Search ADS PubMed  53. Dewing P, Boulware MI, Sinchak K, Christensen A, Mermelstein PG, Micevych P. Membrane estrogen receptor-alpha interactions with metabotropic glutamate receptor 1a modulate female sexual receptivity in rats. J Neurosci . 2007; 27( 35): 9294– 9300. Google Scholar CrossRef Search ADS PubMed  54. Boulware MI, Heisler JD, Frick KM. The memory-enhancing effects of hippocampal estrogen receptor activation involve metabotropic glutamate receptor signaling. J Neurosci . 2013; 33( 38): 15184– 15194. Google Scholar CrossRef Search ADS PubMed  55. Balthazart J, Ball GF. Is brain estradiol a hormone or a neurotransmitter? Trends Neurosci . 2006; 29( 5): 241– 249. Google Scholar CrossRef Search ADS PubMed  56. Balthazart J, Cornil CA, Taziaux M, Charlier TD, Baillien M, Ball GF. Rapid changes in production and behavioral action of estrogens. Neuroscience . 2006; 138( 3): 783– 791. Google Scholar CrossRef Search ADS PubMed  57. Kretz O, Fester L, Wehrenberg U, Zhou L, Brauckmann S, Zhao S, Prange-Kiel J, Naumann T, Jarry H, Frotscher M, Rune GM. Hippocampal synapses depend on hippocampal estrogen synthesis. J Neurosci . 2004; 24( 26): 5913– 5921. Google Scholar CrossRef Search ADS PubMed  58. Fester L, Zhou L, Bütow A, Huber C, von Lossow R, Prange-Kiel J, Jarry H, Rune GM. Cholesterol-promoted synaptogenesis requires the conversion of cholesterol to estradiol in the hippocampus. Hippocampus . 2009; 19( 8): 692– 705. Google Scholar CrossRef Search ADS PubMed  59. Srivastava DP, Waters EM, Mermelstein PG, Kramár EA, Shors TJ, Liu F. Rapid estrogen signaling in the brain: implications for the fine-tuning of neuronal circuitry. J Neurosci . 2011; 31( 45): 16056– 16063. Google Scholar CrossRef Search ADS PubMed  60. Peterson BM, Mermelstein PG, Meisel RL. Estradiol mediates dendritic spine plasticity in the nucleus accumbens core through activation of mGluR5. Brain Struct Funct . 2015; 220( 4): 2415– 2422. Google Scholar CrossRef Search ADS PubMed  61. Santen RJ, Boucher AE, Santner SJ, Henderson IC, Harvey H, Lipton A. Inhibition of aromatase as treatment of breast carcinoma in postmenopausal women. J Lab Clin Med . 1987; 109( 3): 278– 289. Google Scholar PubMed  62. Bloedel JR, Bracha V. Duality of cerebellar motor and cognitive functions. Int Rev Neurobiol . 1997; 41: 613– 634. Google Scholar CrossRef Search ADS PubMed  63. Strick PL, Dum RP, Fiez JA. Cerebellum and nonmotor function. Annu Rev Neurosci . 2009; 32( 1): 413– 434. Google Scholar CrossRef Search ADS PubMed  64. Galliano E, De Zeeuw CI. Questioning the cerebellar doctrine. Prog Brain Res . 2014; 210: 59– 77. Google Scholar CrossRef Search ADS PubMed  65. Rapkin AJ, Berman SM, Mandelkern MA, Silverman DH, Morgan M, London ED. Neuroimaging evidence of cerebellar involvement in premenstrual dysphoric disorder. Biol Psychiatry . 2011; 69( 4): 374– 380. Google Scholar CrossRef Search ADS PubMed  66. Day R, Ganz PA, Costantino JP, Cronin WM, Wickerham DL, Fisher B. Health-related quality of life and tamoxifen in breast cancer prevention: a report from the National Surgical Adjuvant Breast and Bowel Project P-1 Study. J Clin Oncol . 1999; 17( 9): 2659– 2669. Google Scholar CrossRef Search ADS PubMed  67. Demissie S, Silliman RA, Lash TL. Adjuvant tamoxifen: predictors of use, side effects, and discontinuation in older women. J Clin Oncol . 2001; 19( 2): 322– 328. Google Scholar CrossRef Search ADS PubMed  68. McCowan C, Shearer J, Donnan PT, Dewar JA, Crilly M, Thompson AM, Fahey TP. Cohort study examining tamoxifen adherence and its relationship to mortality in women with breast cancer. Br J Cancer . 2008; 99( 11): 1763– 1768. Google Scholar CrossRef Search ADS PubMed  69. Hershman DL, Shao T, Kushi LH, Buono D, Tsai WY, Fehrenbacher L, Kwan M, Gomez SL, Neugut AI. Early discontinuation and non-adherence to adjuvant hormonal therapy are associated with increased mortality in women with breast cancer. Breast Cancer Res Treat . 2011; 126( 2): 529– 537. Google Scholar CrossRef Search ADS PubMed  70. Lin JH, Zhang SM, Manson JE. Predicting adherence to tamoxifen for breast cancer adjuvant therapy and prevention. Cancer Prev Res (Phila) . 2011; 4( 9): 1360– 1365. Google Scholar CrossRef Search ADS PubMed  71. Andreescu CE, Milojkovic BA, Haasdijk ED, Kramer P, De Jong FH, Krust A, De Zeeuw CI, De Jeu MT. Estradiol improves cerebellar memory formation by activating estrogen receptor beta. J Neurosci . 2007; 27( 40): 10832– 10839. Google Scholar CrossRef Search ADS PubMed  72. Sierra A, Azcoitia I, Garcia-Segura L. Endogenous estrogen formation is neuroprotective in model of cerebellar ataxia. Endocrine . 2003; 21( 1): 43– 51. Google Scholar CrossRef Search ADS PubMed  73. Richardson TE, Yang SH, Wen Y, Simpkins JW. Estrogen protection in Friedreich’s ataxia skin fibroblasts. Endocrinology . 2011; 152( 7): 2742– 2749. Google Scholar CrossRef Search ADS PubMed  74. Ghidoni R, Boccardi M, Benussi L, Testa C, Villa A, Pievani M, Gigola L, Sabattoli F, Barbiero L, Frisoni GB, Binetti G. Effects of estrogens on cognition and brain morphology: involvement of the cerebellum. Maturitas . 2006; 54( 3): 222– 228. Google Scholar CrossRef Search ADS PubMed  75. Boccardi M, Ghidoni R, Govoni S, Testa C, Benussi L, Bonetti M, Binetti G, Frisoni GB. Effects of hormone therapy on brain morphology of healthy postmenopausal women: a voxel-based morphometry study. Menopause . 2006; 13( 4): 584– 591. Google Scholar CrossRef Search ADS PubMed  76. Hammar ML, Lindgren R, Berg GE, Möller CG, Niklasson MK. Effects of hormonal replacement therapy on the postural balance among postmenopausal women. Obstet Gynecol . 1996; 88( 6): 955– 960. Google Scholar CrossRef Search ADS PubMed  77. Jung ME, Yang SH, Brun-Zinkernagel AM, Simpkins JW. Estradiol protects against cerebellar damage and motor deficit in ethanol-withdrawn rats. Alcohol . 2002; 26( 2): 83– 93. Google Scholar CrossRef Search ADS PubMed  78. Weill-Engerer S, David JP, Sazdovitch V, Liere P, Schumacher M, Delacourte A, Baulieu EE, Akwa Y. In vitro metabolism of dehydroepiandrosterone (DHEA) to 7alpha-hydroxy-DHEA and delta5-androstene-3beta,17beta-diol in specific regions of the aging brain from Alzheimer’s and non-demented patients. Brain Res . 2003; 969( 1-2): 117– 125. 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Local Estrogen Synthesis Regulates Parallel Fiber–Purkinje Cell Neurotransmission Within the Cerebellar Cortex

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Endocrine Society
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Copyright © 2018 Endocrine Society
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0013-7227
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1945-7170
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10.1210/en.2018-00039
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Abstract

Abstract Estrogens affect cerebellar activity and cerebellum-based behaviors. Within the adult rodent cerebellum, the best-characterized action of estradiol is to enhance glutamatergic signaling. However, the mechanisms by which estradiol promotes glutamatergic neurotransmission remain unknown. Within the mouse cerebellum, we found that estrogen receptor activation of metabotropic glutamate receptor type 1a strongly enhances neurotransmission at the parallel fiber–Purkinje cell synapse. The blockade of local estrogen synthesis within the cerebellum results in a diminution of glutamatergic neurotransmission. Correspondingly, decreased estrogen availability via gonadectomy or blockade of aromatase activity negatively affects locomotor performance. These data indicate that locally derived, and not just gonad-derived, estrogens affect cerebellar physiology and function. In addition, estrogens were found to facilitate parallel fiber–Purkinje cell synaptic transmission in both sexes. As such, the actions of estradiol to support cerebellar neurotransmission and cerebellum-based behaviors might be fundamental to understanding the normal processing of activity within the cerebellar cortex. For decades, estrogens have been known to affect cerebellar function and development in both male and female animals (1–3). Early in rodent development, estrogens influence cerebellar synaptic connectivity and other aspects of its neuroanatomical organization (4–6). Within the adult, estrogens enhance synaptic efficacy and augment cerebellum-mediated behaviors (7–10). The clinical deficits occurring after the administration of estrogen receptor antagonists and aromatase inhibitors in humans also suggest a central role of estradiol in cerebellar function (11–16). Additionally, numerous studies have described neuroprotective effects of estrogens within this brain region (17–20). However, despite all this evidence, we lack a clear understanding regarding the mechanisms by which estrogens exert influence on the cerebellum. Recent findings have suggested that in addition to the activation of nuclear hormone receptors, estrogens affect nervous system function via multiple signaling pathways that are initiated at the cellular membrane (21, 22). Also, peripheral estrogens might not be the sole source of ligand, because evidence from both human and animal studies has shown that estradiol is synthesized locally within the cerebellum (4, 6, 23, 24). Through technological advancement of in vivo imaging studies (25, 26), in conjunction with both established electrophysiological and behavioral techniques, we have reexamined estrogen action within the rodent cerebellum. We have defined a mechanism by which estrogens support normal cerebellar function. In both males and females, estrogen receptor activation supports metabotropic glutamate receptor type 1a (mGluR1a) neurotransmission, which is required for proper parallel fiber (PF)–Purkinje cell synaptic function. Moreover, activation of cerebellar estrogen receptors is at least partially mediated by local estrogen synthesis. Consequently, estrogen depletion results in deficits in locomotor activity, demonstrating the physiological importance of this hormone for normal behavior. Materials and Methods Animals For the mouse experiments at the University of Minnesota, the Institutional Animal Care and Use Committee approved the procedures. Intact and gonadectomized mice (strain, Friend Virus B; age, 3 to 4 months) arrived from Charles River Laboratories (Wilmington, MA). The mice were housed in groups of four (separated by sex) in plastic cages and maintained in a specific pathogen-free colony room. The animal room was maintained at a controlled temperature with a 12-hour light/dark schedule. Food and water were available ad libitum. Experimental procedures began 1 week after acclimation. Experiments in rats were performed at Nanjing University and were in compliance with the U.S. National Institutes of Health Guide for the Care and Use of Laboratory Animals. Patch clamp experiments used male and female rats (Sprague-Dawley; Experimental Animal Center of Nanjing Medical University, Nanjing, China) aged 12 to 16 days. The younger age of the rats was necessary to obtain stable patch clamp recordings. The data are presented as the mean ± standard error of the mean throughout. DigiGait™ acquisition and analysis The mice were given a subcutaneous osmotic minipump implant under isoflurane anesthesia (1.5% to 2%). To prepare for the midscapular incision, the fur was removed, and the area was disinfected with betadine. A midscapular incision was made through the skin, and a subcutaneous pocket was created by opening and closing a hemostat between the skin and muscle and spreading the subcutaneous tissues apart. ALZET osmotic minipumps (model no. 2002; Durect Corporation, Cupertino, CA) were filled under sterile conditions with either the aromatase inhibitor fadrozole (Fad; catalog no. F3806; Sigma-Aldrich, Burlington, MA) in 0.9% saline (0.5 mg/kg/d) or 0.9% saline as per the manufacturer’s instructions. A filled osmotic minipump was inserted into the pocket, with the flow modulator of the pump pointing away from the incision. The incision was closed using wound clips. One week after surgery, the mice were tested for differences in locomotor function. The DigiGait™ System (Mouse Specifics, Inc., Framingham, MA) performs gait analysis of rodents over a range of walking and running speeds by monitoring an animal’s gait continuously via ventral plane images taken through a transparent motorized treadmill at a rate of 80 frames/s. These images generate digital paw prints and a dynamic gait signal for each of the four limbs and quantifies both spatial and temporal indexes of gait. The mice were tested at a “running” speed of 25 cm/s. Videos (5 to 7 seconds) of each mouse were analyzed using the DigiGait™ software, version 12.4, to automatically calculate the values for the gait parameters. The effects were analyzed through two-way analysis of variance (ANOVA), followed by Tukey honest significant difference. In vivo flavoprotein optical imaging and field potential recordings Optical imaging was performed as previously described (27). The mice were anesthetized with urethane (intraperitoneal injection of 1.2 mg/kg urethane, supplemented with 0.3 mg/kg urethane as needed) and mechanically ventilated; the core temperature was maintained. Each mouse was placed in a stereotaxic frame, crus I and II of the cerebellar cortex were exposed, and the dura was removed. An acrylic chamber was constructed around the exposed folia and filled with normal Ringer solution. The mouse and stereotaxic frame were placed on a large stage with precision x and y translation. Flavoprotein-based autofluorescence optical imaging was performed using Nikon epifluorescence optics, with an excitation band-pass filter of 420 to 490 nm, a 500-nm dichroic mirror, and a long-pass filter of >515 nm. The camera was focused just below the surface of the cerebellar cortex, and parallel fibers were activated by a tungsten microelectrode placed just into the molecular layer. The basic imaging paradigm consisted of collecting a time series of 200-ms images before, during, and after parallel fiber (PF) stimulation (a train of 10 pulses at 100 Hz of 200 μA and 100 μs). Having obtained a series of images, the optical response was determined by subtracting the average of the first 20 background images (control images before PF stimulation) from each image in the series acquired to generate a series of “difference” images. These difference images were then divided by the control average on a pixel-by-pixel basis, in which the intensity value of each pixel reflects the change in fluorescence intensity relative to the average of the control frames (ΔF/F). To quantify the response to PF stimulation, a region of interest (ROI), defined by the evoked beam or long-term patches, was visually determined, and the same ROI was used throughout an experiment to quantify any changes in the response. For the beam, the response to the parallel fiber stimulation consisted of an initial increase in fluorescence (light phase) that is tightly coupled to the strength of the stimulation (25). Therefore, the analysis was restricted to the light phase, averaging the five frames centered on the peak amplitude of the light phase to obtain the average ΔF/F within the ROI. The long-latency patches occurred ∼25 to 30 seconds after PF stimulation, an ROI was defined for each patch, and the 25 frames were averaged around the peak to obtain the amplitude (27). Beam and patch intensities during the baseline measurements were compared with those occurring after 50 to 65 minutes of bath-applied drug (or vehicle). The effects were analyzed using a paired Student t test. For the experiments using the metabotropic glutamate receptor (mGluR) 1 antagonist JNJ 16259685 (JNJ; 1 μM) and the estrogen receptor antagonist ICI 182,780 (ICI; 1 μM), fluorescence was measured first for 30 minutes after JNJ treatment and then 30 minutes later in the presence of JNJ and ICI. The effects were analyzed via an ANOVA (within-subject design with repeated measures), followed by a post hoc Bonferroni test. Electrophysiology Field potential recordings Field potential recordings of the responses to PF stimulation provided an electrophysiological assessment of the pre- vs postsynaptic effects of blocking estrogen receptors in the cerebellum of mice. Using established protocols (27), the field potentials in the molecular layer were recorded using glass microelectrodes (2M NaCl, 2 to 5 MΩ), digitized at 25,000 Hz and averaged (responses to 16 single PF stimuli at 1 Hz). The P1/N1 component is a measure of the presynaptic response, and the N2 component is a measure of the postsynaptic response (25, 27, 28). The field potentials were monitored every 3 to 4 minutes for 30 minutes before and for 65 minutes during the drug applications. The field potential amplitudes during the baseline measurements were compared with those after 50 to 65 minutes of bath-applied drugs. The effects of ICI and Fad on the P1/N1 and N2 components were analyzed using ANOVA (within-subject design) followed by a post hoc Bonferroni test. Whole-cell patch clamp Coronal cerebellar slices (400-μm thick) were prepared with a vibroslicer (VT 1200 S; Leica, Germany) guided by the rat brain atlas (29). The slices were incubated in artificial cerebrospinal fluid (ACSF; composition: 124 mM NaCl, 2.5 mM KCl, 1.25 mM NaH2PO4, 1.3 mM MgSO4, 26 mM NaHCO3, 2 mM CaCl2, and 10 mM d-glucose) equilibrated with 95% oxygen and 5% carbon dioxide at 35° ± 0.5°C for ≥1 hour and then maintained at room temperature. During recording sessions, the slices were transferred to a submersion chamber and continuously perfused with 95% oxygen and 5% carbon dioxide–oxygenated ACSF at a rate of 2 mL/min maintained at room temperature. Whole-cell patch recordings were performed as in previous reports (30, 31) on cerebellar cortical Purkinje neurons with borosilicate glass pipettes (3 to 5 MΩ) filled with an internal solution (composition: 140 mM K-methylsulfate, 7 mM KCl, 2 mM MgCl2, 10 mM HEPES, 0.1 mM EGTA, 4 mM Na2-ATP, 0.4 mM GTP-Tris, adjusted to pH 7.25, with 1 M KOH). During the recording sessions, Purkinje neurons were visualized with an Olympus BX51WI microscope. Patch-clamp recordings were acquired with an Axopatch-700B amplifier (Molecular Devices, Sunnyvale, CA). Data capture and analysis was performed using a Digidata-1440A interface and pClamp, version 10.2, software (Molecular Devices). Neurons were held at a membrane potential of −70 mV and characterized by injection of a rectangular voltage pulse (5 mV, 50 ms) to monitor the whole-cell membrane capacitance, series resistance, and membrane resistance. Neurons were excluded from the study if the series resistance was not stable or exceeded 20 MΩ. The slices were bathed in the pan-specific group I mGluR agonist (S)-3,5-dihydroxyphenylglycine (DHPG; 100 μM; Tocris, Minneapolis, MN) to induce an inward current by specific activation of mGluR on the recorded Purkinje neurons. Next, the slices were perfused with ACSF containing 17β-estradiol (1 nM; Sigma-Aldrich) for 20 minutes to observe the effect of estradiol on the mGluR-specific current. Afterward, the actions of estradiol were tested in the presence of the mGluR1-specific antagonist CPCCOEt (125 μM; Tocris) to assess whether the effect of estradiol was dependent on mGluR1. CPCCOEt was used instead of JNJ in these experiments, because the JNJ drug was unavailable to the experimenters at the time. In addition to the pharmacologically induced mGluR current, we determined the effect of estradiol on the slow excitatory postsynaptic currents (sEPSCs) and mGluR excitatory postsynaptic currents (EPSCs) evoked by PF stimulation. A concentric bipolar electrode (CB ARC75; FHC Inc.) was positioned in the molecular layer in the vicinity of the recorded neuron to stimulate the PFs. PFs were activated by applying a train of 10 pulses (20 to 200 μA, 300 μs) at 100 Hz to the electrode through a programmable stimulator (Master-9; A.M.P.I., Jerusalem Israel). The γ-aminobutyric acid A receptor antagonist SR95531 (20 μM) and the glutamate transporter blocker DL-TBOA (50 μM) were applied to inhibit inhibitory synaptic transmission and obtain apparent sEPSCs. The effects of 17β-estradiol (1 nM) on the PF-evoked sEPSCs in the absence and presence of CPCCOEt (125 μM) were evaluated. For whole-cell patch clamp experiments, data were analyzed via a paired Student t test. Estrogen concentration measurements Extraction of estradiol from cerebellar cortex homogenate was adapted from previously reported methods (32). Cerebellar tissue was maintained at −80°C until the day of extraction. Homogenates [one-quarter fraction of tissue in 1 mL of 0.1 M phosphate buffer (PB)] were first liquid extracted with 2 mL of diethyl ether three times in series for maximum yield. After resuspension in 0.1 M PB, the samples were solid phase extracted on C18 columns (Empore) with an elution using 100% MeOH (high-performance liquid chromatography grade). The estradiol concentrations were then measured using a validated enzyme-linked immunosorbent assay (catalog no. 582251; Cayman Chemical, Ann Arbor, MI). The estradiol content measurements from the assay were corrected based on the wet weight (mg) of each cerebellar hemisphere. All samples were detectable above blanks (0.1 M PB with no brain content) with 78.5% extraction efficiency and standard curve R2 at 98%. Results The effect of estrogen depletion on cerebellar functioning was assessed via analysis of locomotor behavior, because the cerebellum is required for normal movement (33) and estrogen increases Purkinje cell activity during locomotion (10, 34). To avoid potential confounders, these behavioral studies solely used male mice, because estrogens are known to affect basal ganglia functioning in females (35), including enhancing sensorimotor performance (36). The behavioral analysis consisted of monitoring treadmill activity via the DigiGait™ System. Both intact and castrated mice were compared. Conspicuously, although the male gonads produce estrogens, they are not the primary hormone produced by the testes. Hence, behavioral changes due to castration alone cannot singly be ascribed to a deficiency in estrogens. Because the brain can also synthesize estrogens directly, as well as can other organs outside the gonads, both intact and castrated mice were implanted with a minipump to systemically deliver either the aromatase inhibitor Fad (0.5 mg/kg/d) or vehicle. Fad administration allowed us to directly attribute alterations in behavior to changes in estradiol availability. Both castration and Fad produced deficits in locomotor performance. Two main behaviors were altered. When examining the ratio in which the rear paws were in a stance position vs swinging forward (Fig. 1A), main effects were found for both castration (F = 16.53; P < 0.01, n = 4 to 10 per group) and Fad (F = 7.08; P < 0.05). Similar results for castration (F = 13.08; P < 0.01) and Fad (F = 5.55, P < 0.05) were found when examining the average time in which both rear paws were on the ground (Fig. 1B). Of particular interest, deficits in these same behaviors were observed in a mouse model of cerebellar ataxia (37). Figure 1. View largeDownload slide Diminution of estrogen signaling adversely affects cerebellum-dependent motor behavior. (A and B) DigiGait™ analysis of locomotor activity in both intact and castrated (CAST) male mice after chronic administration of Fad or vehicle. Both castration and Fad decreased locomotor performance when quantifying the ratio of stance to swing of the (A) rear paw and (B) shared rear paw stance time (*P < 0.05; **P < 0.01). Data presented as mean ± standard error of the mean. Figure 1. View largeDownload slide Diminution of estrogen signaling adversely affects cerebellum-dependent motor behavior. (A and B) DigiGait™ analysis of locomotor activity in both intact and castrated (CAST) male mice after chronic administration of Fad or vehicle. Both castration and Fad decreased locomotor performance when quantifying the ratio of stance to swing of the (A) rear paw and (B) shared rear paw stance time (*P < 0.05; **P < 0.01). Data presented as mean ± standard error of the mean. In attempt to uncover the mechanism by which estradiol affects cerebellar functioning, we turned to activity-dependent optical imaging. Using this technique, we were able to access, in vivo, alterations in cerebellar synaptic signaling. Specifically, changes in the endogenous fluorescence of mitochondrial flavoproteins were used to monitor PF–Purkinje cell synaptic transmission in the anesthetized mouse (Fig. 2A). The PFs are the bifurcated axons of the granule cells and create 100,000 to 200,000 glutamatergic synapses on each postsynaptic Purkinje cell (28). Furthermore, estradiol supports glutamatergic neurotransmission in the cerebellum (7–10). PFs were stimulated with a train of 10 pulses (200 μA, 100 μs) at 100 Hz using a tungsten microelectrode. This stimulation protocol produces two distinct patterns of fluorescent activity, both dependent on glutamatergic neurotransmission. The initial beam-like increase in flavoprotein fluorescence in response to PF stimulation is due to the postsynaptic activation of Purkinje cells and is principally dependent on AMPA receptors (AMPARs), with a smaller contribution from the group I mGluR, mGluR1a (25, 38). On the decay of the beam fluorescent signal, a second fluorescent signal arises, specifically, patches of increased fluorescent activity, driven principally by activation of mGluR1a (27). Figure 2. View largeDownload slide Estrogen receptor activity supports glutamatergic neurotransmission between cerebellar granule cells and Purkinje neurons. (A) Illustration of the experimental setup. (Left) The electrode activates granule cell PFs, initiating glutamatergic neurotransmission across multiple Purkinje neurons. (Middle) PF stimulation evokes an initial beam of fluorescent activity that is primarily dependent on AMPAR activation with a smaller group I mGluR-dependent component, followed several seconds later by (Right) several patches solely reliant on group I mGluR activation. (B) Paired bright-field and ΔF/F images of an exposed cerebellum in which PF activation was initiated with a stimulating electrode (lower right-hand corner of the bright-field image). Shown are the ΔF/F images of the evoked beam and patch responses before and after administration of the estrogen receptor antagonist ICI (1 μM). (C and D) Within 30 minutes of bath application directly to the cerebellum, ICI produced a decrease in both beam and patch responses (**P < 0.01). (E) Paired bright-field and ΔF/F images of a cerebellum exposed to the GPR30 agonist G1 (1 μM). (F and G) G1 had no effect on either the beam or the patch responses. Data presented as mean ± standard error of the mean. Figure 2. View largeDownload slide Estrogen receptor activity supports glutamatergic neurotransmission between cerebellar granule cells and Purkinje neurons. (A) Illustration of the experimental setup. (Left) The electrode activates granule cell PFs, initiating glutamatergic neurotransmission across multiple Purkinje neurons. (Middle) PF stimulation evokes an initial beam of fluorescent activity that is primarily dependent on AMPAR activation with a smaller group I mGluR-dependent component, followed several seconds later by (Right) several patches solely reliant on group I mGluR activation. (B) Paired bright-field and ΔF/F images of an exposed cerebellum in which PF activation was initiated with a stimulating electrode (lower right-hand corner of the bright-field image). Shown are the ΔF/F images of the evoked beam and patch responses before and after administration of the estrogen receptor antagonist ICI (1 μM). (C and D) Within 30 minutes of bath application directly to the cerebellum, ICI produced a decrease in both beam and patch responses (**P < 0.01). (E) Paired bright-field and ΔF/F images of a cerebellum exposed to the GPR30 agonist G1 (1 μM). (F and G) G1 had no effect on either the beam or the patch responses. Data presented as mean ± standard error of the mean. To determine the role of estrogen receptor signaling, measurements of beam and patch signal intensity were compared before and after administration of the estrogen receptor antagonist ICI (1 μM) to the ACSF-filled optical chamber (Fig. 2B). In cerebella from gonadally intact male mice, ICI produced a statistically significant decrease in both the beam (T = 4.84; P < 0.01; n = 5; Fig. 2C) and patch (T = 12.28; P < 0.01; Fig. 2D) fluorescent responses. In contrast, administration of vehicle to the optical chamber had no effect on either measure (n = 4; data not shown). To verify this was not a male-specific effect, the same measurements were obtained in the cerebella of ovariectomized female animals (n = 3), where ICI produced a statistically significant decrease in beam (control, 1.89% ± 0.05% vs ICI, 1.38% ± 0.03% ΔF/F; T = 5.54; P < 0.01) and patch (control, 1.29% ± 0.15% vs ICI, 0.49% ± 0.16% ΔF/F; T = 2.19; P < 0.05) fluorescence. In addition to antagonism of classic estrogen receptors, ICI has also been demonstrated to activate G protein–coupled receptor homolog (GPR30) (39). Thus, to ascertain whether the effect of ICI could be attributed to this alternative receptor, we applied the GPR30 agonist G1 (1 μM) directly to the optical chamber in five male mice (Fig. 2E). G1 was found to have no effect on either the beam (T = 1.43; P = NS; Fig. 2F) or patch (T = 1.48; P = NS; Fig. 2G) response. Decreased activity in both the beam and the patch response by ICI indicated either an estrogen-mediated effect on mGluR signaling alone or an action on both mGluRs and AMPARs. To further explore how estrogen receptor activation is critical for the maintenance of synaptic transmission between PFs and Purkinje cells, the effect of ICI in the presence of the mGluR1a antagonist JNJ (1 μM) was examined in male mice. As expected, JNJ by itself produced a small, but statistically significant, decrease in beam amplitude (F = 9.26; P < 0.01; n = 5; Fig. 3A and 3B), with concurrent elimination of the patch response (F = 15.31; P < 0.01; Fig. 3C). However, ICI produced no additional effect in the presence of JNJ. These data indicate an effect of estrogen dependent on mGluR1a without affecting AMPAR signaling. Although it is well established that in the female rodent nervous system, estradiol activation of membrane-localized estrogen receptors leads to stimulation of group I mGluRs (40), to the best of our knowledge, these are the first experimental data from male rodents to indicate a brain region in the male that responds similarly. These data are also consistent with previous observations in the male quail brain (41). Figure 3. View largeDownload slide Estrogen receptor regulation of PF–Purkinje cell neurotransmission is dependent on group I mGluRs. (A) Paired bright-field and ΔF/F images of an exposed cerebellum in which PF stimulation resulted in beam and patch responses. Baseline fluorescent responses were compared with the responses when the cerebellum was treated first with the group I mGluR antagonist JNJ (1 μM), followed by the combination of JNJ and ICI. (B and C) The effect of estrogen receptor antagonism on the beam and patch responses to PF stimulation was eliminated after inhibition of group I mGluRs. The differences in the results of both treatment groups compared with baseline were statistically significant (**P < 0.01) but were not substantially different from each other. Data presented as mean ± standard error of the mean. Figure 3. View largeDownload slide Estrogen receptor regulation of PF–Purkinje cell neurotransmission is dependent on group I mGluRs. (A) Paired bright-field and ΔF/F images of an exposed cerebellum in which PF stimulation resulted in beam and patch responses. Baseline fluorescent responses were compared with the responses when the cerebellum was treated first with the group I mGluR antagonist JNJ (1 μM), followed by the combination of JNJ and ICI. (B and C) The effect of estrogen receptor antagonism on the beam and patch responses to PF stimulation was eliminated after inhibition of group I mGluRs. The differences in the results of both treatment groups compared with baseline were statistically significant (**P < 0.01) but were not substantially different from each other. Data presented as mean ± standard error of the mean. The reduction of mGluR1a activation after ICI treatment could be due to alterations in pre- and/or postsynaptic activity of the PF synapse. To distinguish between these possibilities, field potential recordings of the response to PF stimulation were compared before and after the ICI addition to the bath (Fig. 4). Analysis of the field potentials (Fig. 4B) indicated a statistically significant effect of ICI (F = 12.35; P < 0.05; n = 4), but only the postsynaptic N2 component significantly decreased (P < 0.01, Bonferroni post hoc) without a substantial effect on the presynaptic P1/N1 component. These experiments were repeated using female mice (n = 4; Fig. 4C). ICI had a similar effect in females (F = 20.24; P < 0.05) as in males, with a reduction only in the N2 (P < 0.01) and not in the P1/N1 component. Figure 4. View largeDownload slide Estrogen receptor signaling is mediated by postsynaptic activity. (A) Field recordings of the responses to parallel fiber stimulation (1 Hz with 100 μA and100 μs pulses; average of 16 trials) before and after application of ICI. (B and C) ICI decreased the postsynaptic N2 component of the electrical recording (**P < 0.01) without affecting the presynaptic P1/N1 components in both (B) male and (C) female mice. Data presented as mean ± standard error of the mean. Figure 4. View largeDownload slide Estrogen receptor signaling is mediated by postsynaptic activity. (A) Field recordings of the responses to parallel fiber stimulation (1 Hz with 100 μA and100 μs pulses; average of 16 trials) before and after application of ICI. (B and C) ICI decreased the postsynaptic N2 component of the electrical recording (**P < 0.01) without affecting the presynaptic P1/N1 components in both (B) male and (C) female mice. Data presented as mean ± standard error of the mean. Previous studies of females demonstrate estrogen receptor activity can lead to either mGluR1a or mGluR5 signaling, dependent on the brain site (42–46). Adult Purkinje neurons typically express only mGluR1a (47, 48), although under some conditions mGluR5 expression is also observed (49). Hence, the next experiment was designed to corroborate previous work by testing whether estrogen signaling in Purkinje neurons was dependent on mGluR1a. In addition, the experimental method was to apply estradiol to determine whether it would enhance glutamatergic signaling in rats, similar in scope to the work previously described by Smith et al. (7–10). Whole-cell recordings of Purkinje neurons from cerebellar slices in both male (n = 3) and female (n = 2) animals examined the acute effect of estradiol (1 nM) on DHPG-induced (100 μM) inward currents (Fig. 5). The inward current produced by the pan-specific group I mGluR agonist was potentiated by estradiol (T = 9.35; P < 0.01, n = 5 recordings, one from each animal). Also, consistent with our hypotheses, application of the mGluR1-specific antagonist CPCCOEt (125 μM) both blocked ∼90% of the total DHPG-induced current and eliminated any effect by the hormone, indicating that in Purkinje cells, estradiol affects cerebellar neurotransmission through modulation of mGluR1a. The residual current insensitive to CPCCOEt might be mGluR5 mediated, because the recordings were taken from animals at an age in which the cerebellum does express mGluR5 (50). Figure 5. View largeDownload slide Estradiol enhanced mGluR1a activity in cerebellar Purkinje neurons. (A) A whole-cell recording from a cerebellar Purkinje neuron. Estradiol (1 nM) potentiated the inward current induced by the group I mGluR1 agonist DHPG (100 μM). Isolation of mGluR5 after bath application of the mGluR1 antagonist CPCCOEt (125 μM) eliminated the effect of estradiol, demonstrating estradiol-mediated enhancement of mGluR1a. (B) Group data demonstrating estradiol-mediated enhancement of DHPG-induced current (**P < 0.01) is dependent on mGluR1a. (C) Estradiol potentiation of sEPSCs is dependent on mGluR1a. A whole-cell recording of sEPSCs in a Purkinje neuron after stimulation of the molecular layer. Estradiol (1 nM) enhancement of the sEPSC was eliminated after administration of the mGluR1 antagonist CPCCOEt. (D) Group data demonstrating estradiol-mediated enhancement of sEPSCs (**P < 0.01) is dependent on mGluR1a. Data presented as mean ± standard error of the mean. Figure 5. View largeDownload slide Estradiol enhanced mGluR1a activity in cerebellar Purkinje neurons. (A) A whole-cell recording from a cerebellar Purkinje neuron. Estradiol (1 nM) potentiated the inward current induced by the group I mGluR1 agonist DHPG (100 μM). Isolation of mGluR5 after bath application of the mGluR1 antagonist CPCCOEt (125 μM) eliminated the effect of estradiol, demonstrating estradiol-mediated enhancement of mGluR1a. (B) Group data demonstrating estradiol-mediated enhancement of DHPG-induced current (**P < 0.01) is dependent on mGluR1a. (C) Estradiol potentiation of sEPSCs is dependent on mGluR1a. A whole-cell recording of sEPSCs in a Purkinje neuron after stimulation of the molecular layer. Estradiol (1 nM) enhancement of the sEPSC was eliminated after administration of the mGluR1 antagonist CPCCOEt. (D) Group data demonstrating estradiol-mediated enhancement of sEPSCs (**P < 0.01) is dependent on mGluR1a. Data presented as mean ± standard error of the mean. To verify estradiol potentiation of mGluR1a signaling, a second whole-cell patch clamp experiment was performed to determine the effect of estradiol on PF-evoked EPSCs in Purkinje cells in the absence or presence of the mGluR1 antagonist CPCCOEt (Fig. 6). To that end, we exploited the fact that group I mGluR signaling can enhance electrically evoked EPSCs (51). Consistent with estradiol specifically affecting mGluR1a signaling, estradiol, in both male and female neurons, enhanced EPSCs under control conditions (T = 6.11; P < 0.01) but had no effect in the presence of CPCCOEt (n = 6 recordings; n = 3 separate animals for each sex). Figure 6. View largeDownload slide Local estrogen synthesis facilitates cerebellar PF–Purkinje cell neurotransmission. (A) Paired bright-field and ΔF/F images of the beam and patch responses to PF simulation in a castrated (CAST) mouse. Optical responses were obtained before and after administration of the aromatase inhibitor Fad (1 μM). (B and C) Within 45 minutes of bath application, Fad produced a decrease in both beam and patch (**P < 0.01) signals. (D) Paired bright-field and ΔF/F images of the beam and patch responses to PF simulation in an ovariectomized (OVX) mouse. (E and F) Similar to castrated mice, Fad produced a decrease in both beam (*P < 0.05) and patch (**P < 0.01) responses in ovariectomized mice. Data presented as mean ± standard error of the mean. Figure 6. View largeDownload slide Local estrogen synthesis facilitates cerebellar PF–Purkinje cell neurotransmission. (A) Paired bright-field and ΔF/F images of the beam and patch responses to PF simulation in a castrated (CAST) mouse. Optical responses were obtained before and after administration of the aromatase inhibitor Fad (1 μM). (B and C) Within 45 minutes of bath application, Fad produced a decrease in both beam and patch (**P < 0.01) signals. (D) Paired bright-field and ΔF/F images of the beam and patch responses to PF simulation in an ovariectomized (OVX) mouse. (E and F) Similar to castrated mice, Fad produced a decrease in both beam (*P < 0.05) and patch (**P < 0.01) responses in ovariectomized mice. Data presented as mean ± standard error of the mean. With estrogen receptor activation of mGluR1a signaling required for maximal PF–Purkinje cell neurotransmission, we sought to revisit whether local estrogen synthesis contributes to this process. Cerebellar fractions from intact male mice contained 5.92 ± 3.29 pg/mg estradiol, which were not different from those of castrated mice (8.81 ± 3.19 pg/mg; n = 8/group; T = 0.63; P = NS; assay blanks were 0.04 pg), indicating sources of estrogen were available to the cerebellum independent of the gonads. Parallel to the interaction observed between castration and Fad treatment on locomotor behavior, we next sought to directly test whether cerebellar neuroestrogen synthesis contributed to PF–Purkinje cell neurotransmission. Thus, beam and patch fluorescence in castrated animals were examined after direct administration of Fad (1 μM) to the cerebellar cortex (Fig. 6A–6C). Fad produced a reduction in both beam (T = 7.21; P < 0.01; n = 5) and patch (T = 4.59; P < .01) responses. However, the effects of Fad were not dependent on castration, because in intact male animals (n = 4), Fad also produced a statistically significant reduction to the beam (control, 0.68% ± 0.09% vs Fad, 0.25% ± 0.05% ΔF/F; T = 9.41; P < 0.01) and patch (control, 0.40% ± 0.09% vs Fad, 0.05% ± 0.03% ΔF/F, T = 5.51; P < 0.01) responses. Finally, similar effects were also observed in the cerebella of ovariectomized animals (Fig. 6D–6F), with Fad producing a reduction in both beam (T = 3.85; P < 0.05) and patch (T = 13.12; P < 0.01) responses (n = 4). Discussion The present data support the conclusion that estrogen signaling is critical for both PF–Purkinje cell synaptic transmission and cerebellum-dependent motoric behaviors. Not only does estrogen regulation of Purkinje neuron mGluR1a signaling appear to be a component of normal cerebellar function, but it also displays no overt sex differences, with many similar effects observed in both males and females. Moreover, the cerebellar cortex appears to be a source of estradiol, furthering the notion that this hormone can act locally as a neuromodulator. As such, estradiol regulation of mGluR1a signaling in support of the PF–Purkinje cell synapse provides an underlying mechanism to understand a decades-long uncertainty regarding estrogen function within the adult cerebellum. The concept that estrogen supports basal cerebellar neurotransmission might seem heretical. However, accumulating evidence has been consistent with this hypothesis. Estrogen enhancement of cerebellar glutamatergic neurotransmission, the expression of aromatase within the cerebellum, and the dysfunction of behaviors attributable to cerebellar performance after administration of aromatase inhibitors are three distinct lines of evidence that support this hypothesis (52). Uncovering estrogen regulation of mGluR1a signaling as a mechanism by which this steroid hormone acts within the cerebellum integrates these previous studies. It should be noted, however, that although our data are consistent with this hypothesis, alternative explanations exist. Owing to the nature of behavioral studies, we cannot unequivocally rule out effects of Fad in other brain regions or the periphery. Second, the level of aromatase inhibition achieved by central administration of fadrozole is unknown. The observed effects could not only be due to a decrease in estradiol but also to an increase in testosterone. Finally, although estrogen receptor coupling to mGluR1a has been demonstrated in various systems (42, 46, 53, 54), estrogen receptor activation could facilitate mGluR1a function through a different pathway. From various experimental systems, we know that estradiol synthesis occurs within the nervous system, can exert effects at the cell membrane in response to a stimulus, and exhibits enzymatic inactivation to terminate signaling. Based on these benchmarks, it has been hypothesized that estradiol can act as a neurotransmitter (55, 56). Our findings regarding the diminution of locomotor behavior and PF–Purkinje cell synaptic transmission after depletion of cerebellar estrogens without directly manipulating the glutamate concentrations within this brain region further support this model. The work by Smith et al. (8, 10) first demonstrated that estrogens enhance glutamatergic neurotransmission in the cerebellum, with a corresponding increase in Purkinje cell activity during locomotor behavior. However, this phenomenon might not be unique to the cerebellum, because estradiol regulation of glutamatergic neurotransmission and connectivity has been reported in several other brain regions (57–60). The decreased locomotor performance of male mice after Fad administration is consistent with data from human studies in which some women administered aromatase inhibitors have exhibited ataxia (61). Importantly, the cerebellum is involved in more than just motor-related behaviors, including language, executive function, attention, working memory, pain, emotion, and addiction (62–64). Estrogen signaling in the cerebellum might influence memory (11, 12), cognition (13–16), and mood (65). It is possible that these and other influences underlie the low adherence rate of patients taking tamoxifen therapy, although a lack of compliance results in a striking increase in the mortality rate (66–70). Through pharmacological manipulations that circumvent the steroid hormone receptor, it might be possible to gain novel therapeutic strategies to inhibit estrogen-responsive cancers with concurrent maintenance of nervous system function. Local estrogen regulation of mGluR signaling in the Purkinje neuron is most likely just one of several actions of estradiol in the cerebellum. Endogenous estradiol affects Purkinje cell dendritic growth, spine density, synaptogenesis, and excitability (4–6) and enhances long-term potentiation and vestibulo-ocular reflexes (71). Cerebellar estrogen signaling also provides neuroprotection from a variety of insults (72, 73). Its supplementation preserves the cerebellar gray matter as a function of aging (74, 75) and might improve locomotor and cognitive function in both disease and aging paradigms (76–78). Future work will need to gain a greater appreciation for the role estradiol plays in maintaining and modulating cerebellar function during development, in the adult, and in disease states. In conclusion, estradiol promotion of the PF–Purkinje cell synapse is an aspect of cerebellar physiology. Furthermore, the cerebellum appears capable of synthesizing estrogens to ensure proper neurotransmission, and deficits in estrogen production appear to adversely affect cerebellar function. Abbreviations: Abbreviations: ACSF artificial cerebrospinal fluid AMPAR AMPA receptor ANOVA analysis of variance DHPG (S)-3,5-dihydroxyphenylglycine EPSC excitatory postsynaptic current Fad fadrozole GPR30 G protein–coupled receptor homolog ICI ICI 182,780 JNJ JNJ 16259685 PF parallel fiber ROI region of interest mGluR metabotropic glutamate receptor mGluR1a metabotropic glutamate receptor type 1a sEPSC slow excitatory postsynaptic current ΔF/F change in fluorescence intensity relative to the average of the control frames Acknowledgments The authors thank Dr. Robert Meisel for his comments and suggestions pertaining to the present work. Financial Support: This work was supported by the National Institute of Neurologic Disorders and Stroke (Grant NS077661 to P.G.M. and T.J.E.; Grants NS062158 and NS18338 to T.J.E.; and Grants NS066179 and NS082179 to L.R.-H.); the National Institute on Drug Abuse (Grants DA035008 and DA041808 to P.G.M.); the National Natural Science Foundation of China (Grants 31330033, 91332124, and 31461163001 to J.-J.W.; and Grants 81671107 and 31471112 to J.-N.Z.); the Specialized Research Fund for the Doctoral Program of Higher Education and Research Grants Council Earmarked Research Grants (Grant 20130091140003 to J.-J.W.); and the Jiangsu Natural Science Foundation (Grant BK2011014 to J.-J.W.). Disclosure Summary: The authors have nothing to disclose. References 1. McEwen BS, Pfaff DW. Factors influencing sex hormone uptake by rat brain regions. I. Effects of neonatal treatment, hypophysectomy, and competing steroid on estradiol uptake. Brain Res . 1970; 21( 1): 1– 16. Google Scholar CrossRef Search ADS PubMed  2. Eleftheriou BE, Desjardins C, Pattison ML. Effects of ovariectomy and ovarian hormones on RNA base ratios in specific rabbit brain areas. J Endocrinol . 1970; 46( 3): 331– 340. Google Scholar CrossRef Search ADS PubMed  3. Greengrass PM, Tonge SR. Suggestions on the pharmacological actions of ethinyloestradiol and progesterone on the control of monoamine metabolism in three regions from the brains of gonadectomized male and female mice and the possible clinical significance. Arch Int Pharmacodyn Ther . 1974; 211( 2): 291– 304. Google Scholar PubMed  4. Sakamoto H, Mezaki Y, Shikimi H, Ukena K, Tsutsui K. Dendritic growth and spine formation in response to estrogen in the developing Purkinje cell. Endocrinology . 2003; 144( 10): 4466– 4477. Google Scholar CrossRef Search ADS PubMed  5. Sasahara K, Shikimi H, Haraguchi S, Sakamoto H, Honda S, Harada N, Tsutsui K. Mode of action and functional significance of estrogen-inducing dendritic growth, spinogenesis, and synaptogenesis in the developing Purkinje cell. J Neurosci . 2007; 27( 28): 7408– 7417. Google Scholar CrossRef Search ADS PubMed  6. Dean SL, Wright CL, Hoffman JF, Wang M, Alger BE, McCarthy MM. Prostaglandin E2 stimulates estradiol synthesis in the cerebellum postnatally with associated effects on Purkinje neuron dendritic arbor and electrophysiological properties. Endocrinology . 2012; 153( 11): 5415– 5427. Google Scholar CrossRef Search ADS PubMed  7. Smith SS. Estrogen administration increases neuronal responses to excitatory amino acids as a long-term effect. Brain Res . 1989; 503( 2): 354– 357. Google Scholar CrossRef Search ADS PubMed  8. Smith SS, Waterhouse BD, Woodward DJ. Locally applied estrogens potentiate glutamate-evoked excitation of cerebellar Purkinje cells. Brain Res . 1988; 475( 2): 272– 282. Google Scholar CrossRef Search ADS PubMed  9. Smith SS, Waterhouse BD, Woodward DJ. Sex steroid effects on extrahypothalamic CNS. I. Estrogen augments neuronal responsiveness to iontophoretically applied glutamate in the cerebellum. Brain Res . 1987; 422( 1): 40– 51. Google Scholar CrossRef Search ADS PubMed  10. Smith SS, Woodward DJ, Chapin JK. Sex steroids modulate motor-correlated increases in cerebellar discharge. Brain Res . 1989; 476( 2): 307– 316. Google Scholar CrossRef Search ADS PubMed  11. Phillips SM, Sherwin BB. Variations in memory function and sex steroid hormones across the menstrual cycle. Psychoneuroendocrinology . 1992; 17( 5): 497– 506. Google Scholar CrossRef Search ADS PubMed  12. Phillips SM, Sherwin BB. Effects of estrogen on memory function in surgically menopausal women. Psychoneuroendocrinology . 1992; 17( 5): 485– 495. Google Scholar CrossRef Search ADS PubMed  13. Rocca WA, Bower JH, Maraganore DM, Ahlskog JE, Grossardt BR, de Andrade M, Melton LJ III. Increased risk of cognitive impairment or dementia in women who underwent oophorectomy before menopause. Neurology . 2007; 69( 11): 1074– 1083. Google Scholar CrossRef Search ADS PubMed  14. Schilder CM, Seynaeve C, Beex LV, Boogerd W, Linn SC, Gundy CM, Huizenga HM, Nortier JW, van de Velde CJ, van Dam FS, Schagen SB. Effects of tamoxifen and exemestane on cognitive functioning of postmenopausal patients with breast cancer: results from the neuropsychological side study of the tamoxifen and exemestane adjuvant multinational trial. J Clin Oncol . 2010; 28( 8): 1294– 1300. Google Scholar CrossRef Search ADS PubMed  15. Schilder CM, Seynaeve C, Linn SC, Boogerd W, Beex LV, Gundy CM, Nortier JW, van de Velde CJ, van Dam FS, Schagen SB. Cognitive functioning of postmenopausal breast cancer patients before adjuvant systemic therapy, and its association with medical and psychological factors. Crit Rev Oncol Hematol . 2010; 76( 2): 133– 141. Google Scholar CrossRef Search ADS PubMed  16. Schilder CM, Seynaeve C, Linn SC, Boogerd W, Gundy CM, Beex LV, van Dam FS, Schagen SB. The impact of different definitions and reference groups on the prevalence of cognitive impairment: a study in postmenopausal breast cancer patients before the start of adjuvant systemic therapy. Psychooncology . 2010; 19( 4): 415– 422. Google Scholar CrossRef Search ADS PubMed  17. Rewal M, Wen Y, Simpkins JW, Jung ME. Ethanol withdrawal reduces cerebellar parvalbumin expression in a manner reversed by estrogens. Neurosci Lett . 2005; 377( 1): 44– 48. Google Scholar CrossRef Search ADS PubMed  18. Mirzatoni A, Spence RD, Naranjo KC, Saldanha CJ, Schlinger BA. Injury-induced regulation of steroidogenic gene expression in the cerebellum. J Neurotrauma . 2010; 27( 10): 1875– 1882. Google Scholar CrossRef Search ADS PubMed  19. Zorrilla Zubilete MA, Guelman LR, Maur DG, Caceres LG, Rios H, Zieher LM, Genaro AM. Partial neuroprotection by 17-β-estradiol in neonatal γ-irradiated rat cerebellum. Neurochem Int . 2011; 58( 3): 273– 280. Google Scholar CrossRef Search ADS PubMed  20. Firozan B, Goudarzi I, Elahdadi Salmani M, Lashkarbolouki T, Rezaei A, Abrari K. Estradiol increases expression of the brain-derived neurotrophic factor after acute administration of ethanol in the neonatal rat cerebellum. Eur J Pharmacol . 2014; 732: 1– 11. Google Scholar CrossRef Search ADS PubMed  21. Mermelstein PG, Micevych PE. Nervous system physiology regulated by membrane estrogen receptors. Rev Neurosci . 2008; 19( 6): 413– 424. Google Scholar CrossRef Search ADS PubMed  22. Kelly MJ, Rønnekleiv OK. Membrane-initiated actions of estradiol that regulate reproduction, energy balance and body temperature. Front Neuroendocrinol . 2012; 33( 4): 376– 387. Google Scholar CrossRef Search ADS PubMed  23. Biegon A, Kim SW, Alexoff DL, Jayne M, Carter P, Hubbard B, King P, Logan J, Muench L, Pareto D, Schlyer D, Shea C, Telang F, Wang GJ, Xu Y, Fowler JS. Unique distribution of aromatase in the human brain: in vivo studies with PET and [N-methyl-11C]vorozole. Synapse . 2010; 64( 11): 801– 807. Google Scholar CrossRef Search ADS PubMed  24. Azcoitia I, Yague JG, Garcia-Segura LM. Estradiol synthesis within the human brain. Neuroscience . 2011; 191: 139– 147. Google Scholar CrossRef Search ADS PubMed  25. Reinert KC, Dunbar RL, Gao W, Chen G, Ebner TJ. Flavoprotein autofluorescence imaging of neuronal activation in the cerebellar cortex in vivo. J Neurophysiol . 2004; 92( 1): 199– 211. Google Scholar CrossRef Search ADS PubMed  26. Reinert KC, Gao W, Chen G, Wang X, Peng YP, Ebner TJ. Cellular and metabolic origins of flavoprotein autofluorescence in the cerebellar cortex in vivo. Cerebellum . 2011; 10( 3): 585– 599. Google Scholar CrossRef Search ADS PubMed  27. Wang X, Chen G, Gao W, Ebner TJ. Parasagittally aligned, mGluR1-dependent patches are evoked at long latencies by parallel fiber stimulation in the mouse cerebellar cortex in vivo. J Neurophysiol . 2011; 105( 4): 1732– 1746. Google Scholar CrossRef Search ADS PubMed  28. Eccles JC. The Cerebellum as a Neuronal Machine. Berlin: Springer-Verlag; 1967. 29. Paxinos G, Watson C. The Rat Brain in Stereotaxic Coordinates . 6th ed. New York: Academic Press. 30. Zhang J, Li B, Yu L, He YC, Li HZ, Zhu JN, Wang JJ. A role for orexin in central vestibular motor control. Neuron . 2011; 69( 4): 793– 804. Google Scholar CrossRef Search ADS PubMed  31. Zhang XY, Yu L, Zhuang QX, Peng SY, Zhu JN, Wang JJ. Postsynaptic mechanisms underlying the excitatory action of histamine on medial vestibular nucleus neurons in rats. Br J Pharmacol . 2013; 170( 1): 156– 169. Google Scholar CrossRef Search ADS PubMed  32. Chao A, Schlinger BA, Remage-Healey L. Combined liquid and solid-phase extraction improves quantification of brain estrogen content. Front Neuroanat . 2011; 5: 57. Google Scholar CrossRef Search ADS PubMed  33. Glickstein M, Strata P, Voogd J. Cerebellum: history. Neuroscience . 2009; 162( 3): 549– 559. Google Scholar CrossRef Search ADS PubMed  34. Smith SS. Sensorimotor-correlated discharge recorded from ensembles of cerebellar Purkinje cells varies across the estrous cycle of the rat. J Neurophysiol . 1995; 74( 3): 1095– 1108. Google Scholar CrossRef Search ADS PubMed  35. Becker JB. Gender differences in dopaminergic function in striatum and nucleus accumbens. Pharmacol Biochem Behav . 1999; 64( 4): 803– 812. Google Scholar CrossRef Search ADS PubMed  36. Becker JB, Snyder PJ, Miller MM, Westgate SA, Jenuwine MJ. The influence of estrous cycle and intrastriatal estradiol on sensorimotor performance in the female rat. Pharmacol Biochem Behav . 1987; 27( 1): 53– 59. Google Scholar CrossRef Search ADS PubMed  37. Du X, Wang J, Zhu H, Rinaldo L, Lamar KM, Palmenberg AC, Hansel C, Gomez CM. Second cistron in CACNA1A gene encodes a transcription factor mediating cerebellar development and SCA6. Cell . 2013; 154( 1): 118– 133. Google Scholar CrossRef Search ADS PubMed  38. Wang X, Chen G, Gao W, Ebner T. Long-term potentiation of the responses to parallel fiber stimulation in mouse cerebellar cortex in vivo. Neuroscience . 2009; 162( 3): 713– 722. Google Scholar CrossRef Search ADS PubMed  39. Filardo EJ, Quinn JA, Bland KI, Frackelton AR Jr. Estrogen-induced activation of Erk-1 and Erk-2 requires the G protein-coupled receptor homolog, GPR30, and occurs via trans-activation of the epidermal growth factor receptor through release of HB-EGF. Mol Endocrinol . 2000; 14( 10): 1649– 1660. Google Scholar CrossRef Search ADS PubMed  40. Meitzen J, Mermelstein PG. Estrogen receptors stimulate brain region specific metabotropic glutamate receptors to rapidly initiate signal transduction pathways. J Chem Neuroanat . 2011; 42( 4): 236– 241. Google Scholar CrossRef Search ADS PubMed  41. Seredynski AL, Balthazart J, Ball GF, Cornil CA. Estrogen receptor β activation rapidly modulates male sexual motivation through the transactivation of metabotropic glutamate receptor 1a. J Neurosci . 2015; 35( 38): 13110– 13123. Google Scholar CrossRef Search ADS PubMed  42. Boulware MI, Weick JP, Becklund BR, Kuo SP, Groth RD, Mermelstein PG. Estradiol activates group I and II metabotropic glutamate receptor signaling, leading to opposing influences on cAMP response element-binding protein. J Neurosci . 2005; 25( 20): 5066– 5078. Google Scholar CrossRef Search ADS PubMed  43. Grove-Strawser D, Boulware MI, Mermelstein PG. Membrane estrogen receptors activate the metabotropic glutamate receptors mGluR5 and mGluR3 to bidirectionally regulate CREB phosphorylation in female rat striatal neurons. Neuroscience . 2010; 170( 4): 1045– 1055. Google Scholar CrossRef Search ADS PubMed  44. Christensen A, Dewing P, Micevych P. Membrane-initiated estradiol signaling induces spinogenesis required for female sexual receptivity. J Neurosci . 2011; 31( 48): 17583– 17589. Google Scholar CrossRef Search ADS PubMed  45. Chaban V, Li J, McDonald JS, Rapkin A, Micevych P. Estradiol attenuates the adenosine triphosphate-induced increase of intracellular calcium through group II metabotropic glutamate receptors in rat dorsal root ganglion neurons. J Neurosci Res . 2011; 89( 11): 1707– 1710. Google Scholar CrossRef Search ADS PubMed  46. Huang GZ, Woolley CS. Estradiol acutely suppresses inhibition in the hippocampus through a sex-specific endocannabinoid and mGluR-dependent mechanism. Neuron . 2012; 74( 5): 801– 808. Google Scholar CrossRef Search ADS PubMed  47. Masu M, Tanabe Y, Tsuchida K, Shigemoto R, Nakanishi S. Sequence and expression of a metabotropic glutamate receptor. Nature . 1991; 349( 6312): 760– 765. Google Scholar CrossRef Search ADS PubMed  48. Baude A, Nusser Z, Roberts JD, Mulvihill E, McIlhinney RA, Somogyi P. The metabotropic glutamate receptor (mGluR1 alpha) is concentrated at perisynaptic membrane of neuronal subpopulations as detected by immunogold reaction. Neuron . 1993; 11( 4): 771– 787. Google Scholar CrossRef Search ADS PubMed  49. Fazio F, Notartomaso S, Aronica E, Storto M, Battaglia G, Vieira E, Gatti S, Bruno V, Biagioni F, Gradini R, Nicoletti F, Di Marco R. Switch in the expression of mGlu1 and mGlu5 metabotropic glutamate receptors in the cerebellum of mice developing experimental autoimmune encephalomyelitis and in autoptic cerebellar samples from patients with multiple sclerosis. Neuropharmacology . 2008; 55( 4): 491– 499. Google Scholar CrossRef Search ADS PubMed  50. Casabona G, Knöpfel T, Kuhn R, Gasparini F, Baumann P, Sortino MA, Copani A, Nicoletti F. Expression and coupling to polyphosphoinositide hydrolysis of group I metabotropic glutamate receptors in early postnatal and adult rat brain. Eur J Neurosci . 1997; 9( 1): 12– 17. Google Scholar CrossRef Search ADS PubMed  51. Dzubay JA, Otis TS. Climbing fiber activation of metabotropic glutamate receptors on cerebellar Purkinje neurons. Neuron . 2002; 36( 6): 1159– 1167. Google Scholar CrossRef Search ADS PubMed  52. Hedges VL, Ebner TJ, Meisel RL, Mermelstein PG. The cerebellum as a target for estrogen action. Front Neuroendocrinol . 2012; 33( 4): 403– 411. Google Scholar CrossRef Search ADS PubMed  53. Dewing P, Boulware MI, Sinchak K, Christensen A, Mermelstein PG, Micevych P. Membrane estrogen receptor-alpha interactions with metabotropic glutamate receptor 1a modulate female sexual receptivity in rats. J Neurosci . 2007; 27( 35): 9294– 9300. Google Scholar CrossRef Search ADS PubMed  54. Boulware MI, Heisler JD, Frick KM. The memory-enhancing effects of hippocampal estrogen receptor activation involve metabotropic glutamate receptor signaling. J Neurosci . 2013; 33( 38): 15184– 15194. Google Scholar CrossRef Search ADS PubMed  55. Balthazart J, Ball GF. Is brain estradiol a hormone or a neurotransmitter? Trends Neurosci . 2006; 29( 5): 241– 249. Google Scholar CrossRef Search ADS PubMed  56. Balthazart J, Cornil CA, Taziaux M, Charlier TD, Baillien M, Ball GF. Rapid changes in production and behavioral action of estrogens. Neuroscience . 2006; 138( 3): 783– 791. Google Scholar CrossRef Search ADS PubMed  57. Kretz O, Fester L, Wehrenberg U, Zhou L, Brauckmann S, Zhao S, Prange-Kiel J, Naumann T, Jarry H, Frotscher M, Rune GM. Hippocampal synapses depend on hippocampal estrogen synthesis. J Neurosci . 2004; 24( 26): 5913– 5921. Google Scholar CrossRef Search ADS PubMed  58. Fester L, Zhou L, Bütow A, Huber C, von Lossow R, Prange-Kiel J, Jarry H, Rune GM. Cholesterol-promoted synaptogenesis requires the conversion of cholesterol to estradiol in the hippocampus. Hippocampus . 2009; 19( 8): 692– 705. Google Scholar CrossRef Search ADS PubMed  59. Srivastava DP, Waters EM, Mermelstein PG, Kramár EA, Shors TJ, Liu F. Rapid estrogen signaling in the brain: implications for the fine-tuning of neuronal circuitry. J Neurosci . 2011; 31( 45): 16056– 16063. Google Scholar CrossRef Search ADS PubMed  60. Peterson BM, Mermelstein PG, Meisel RL. Estradiol mediates dendritic spine plasticity in the nucleus accumbens core through activation of mGluR5. Brain Struct Funct . 2015; 220( 4): 2415– 2422. Google Scholar CrossRef Search ADS PubMed  61. Santen RJ, Boucher AE, Santner SJ, Henderson IC, Harvey H, Lipton A. Inhibition of aromatase as treatment of breast carcinoma in postmenopausal women. J Lab Clin Med . 1987; 109( 3): 278– 289. Google Scholar PubMed  62. Bloedel JR, Bracha V. Duality of cerebellar motor and cognitive functions. Int Rev Neurobiol . 1997; 41: 613– 634. Google Scholar CrossRef Search ADS PubMed  63. Strick PL, Dum RP, Fiez JA. Cerebellum and nonmotor function. Annu Rev Neurosci . 2009; 32( 1): 413– 434. Google Scholar CrossRef Search ADS PubMed  64. Galliano E, De Zeeuw CI. Questioning the cerebellar doctrine. Prog Brain Res . 2014; 210: 59– 77. Google Scholar CrossRef Search ADS PubMed  65. Rapkin AJ, Berman SM, Mandelkern MA, Silverman DH, Morgan M, London ED. Neuroimaging evidence of cerebellar involvement in premenstrual dysphoric disorder. Biol Psychiatry . 2011; 69( 4): 374– 380. Google Scholar CrossRef Search ADS PubMed  66. Day R, Ganz PA, Costantino JP, Cronin WM, Wickerham DL, Fisher B. Health-related quality of life and tamoxifen in breast cancer prevention: a report from the National Surgical Adjuvant Breast and Bowel Project P-1 Study. J Clin Oncol . 1999; 17( 9): 2659– 2669. Google Scholar CrossRef Search ADS PubMed  67. Demissie S, Silliman RA, Lash TL. Adjuvant tamoxifen: predictors of use, side effects, and discontinuation in older women. J Clin Oncol . 2001; 19( 2): 322– 328. Google Scholar CrossRef Search ADS PubMed  68. McCowan C, Shearer J, Donnan PT, Dewar JA, Crilly M, Thompson AM, Fahey TP. Cohort study examining tamoxifen adherence and its relationship to mortality in women with breast cancer. Br J Cancer . 2008; 99( 11): 1763– 1768. Google Scholar CrossRef Search ADS PubMed  69. Hershman DL, Shao T, Kushi LH, Buono D, Tsai WY, Fehrenbacher L, Kwan M, Gomez SL, Neugut AI. Early discontinuation and non-adherence to adjuvant hormonal therapy are associated with increased mortality in women with breast cancer. Breast Cancer Res Treat . 2011; 126( 2): 529– 537. Google Scholar CrossRef Search ADS PubMed  70. Lin JH, Zhang SM, Manson JE. Predicting adherence to tamoxifen for breast cancer adjuvant therapy and prevention. Cancer Prev Res (Phila) . 2011; 4( 9): 1360– 1365. Google Scholar CrossRef Search ADS PubMed  71. Andreescu CE, Milojkovic BA, Haasdijk ED, Kramer P, De Jong FH, Krust A, De Zeeuw CI, De Jeu MT. Estradiol improves cerebellar memory formation by activating estrogen receptor beta. J Neurosci . 2007; 27( 40): 10832– 10839. Google Scholar CrossRef Search ADS PubMed  72. Sierra A, Azcoitia I, Garcia-Segura L. Endogenous estrogen formation is neuroprotective in model of cerebellar ataxia. Endocrine . 2003; 21( 1): 43– 51. Google Scholar CrossRef Search ADS PubMed  73. Richardson TE, Yang SH, Wen Y, Simpkins JW. Estrogen protection in Friedreich’s ataxia skin fibroblasts. Endocrinology . 2011; 152( 7): 2742– 2749. Google Scholar CrossRef Search ADS PubMed  74. Ghidoni R, Boccardi M, Benussi L, Testa C, Villa A, Pievani M, Gigola L, Sabattoli F, Barbiero L, Frisoni GB, Binetti G. Effects of estrogens on cognition and brain morphology: involvement of the cerebellum. Maturitas . 2006; 54( 3): 222– 228. Google Scholar CrossRef Search ADS PubMed  75. Boccardi M, Ghidoni R, Govoni S, Testa C, Benussi L, Bonetti M, Binetti G, Frisoni GB. Effects of hormone therapy on brain morphology of healthy postmenopausal women: a voxel-based morphometry study. Menopause . 2006; 13( 4): 584– 591. Google Scholar CrossRef Search ADS PubMed  76. Hammar ML, Lindgren R, Berg GE, Möller CG, Niklasson MK. Effects of hormonal replacement therapy on the postural balance among postmenopausal women. Obstet Gynecol . 1996; 88( 6): 955– 960. Google Scholar CrossRef Search ADS PubMed  77. Jung ME, Yang SH, Brun-Zinkernagel AM, Simpkins JW. Estradiol protects against cerebellar damage and motor deficit in ethanol-withdrawn rats. Alcohol . 2002; 26( 2): 83– 93. Google Scholar CrossRef Search ADS PubMed  78. Weill-Engerer S, David JP, Sazdovitch V, Liere P, Schumacher M, Delacourte A, Baulieu EE, Akwa Y. In vitro metabolism of dehydroepiandrosterone (DHEA) to 7alpha-hydroxy-DHEA and delta5-androstene-3beta,17beta-diol in specific regions of the aging brain from Alzheimer’s and non-demented patients. Brain Res . 2003; 969( 1-2): 117– 125. Google Scholar CrossRef Search ADS PubMed  Copyright © 2018 Endocrine Society

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Published: Mar 1, 2018

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