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REPORT
Department of Cell Biology and Neuroscience, University of California, Riverside, California
Submitted 2 December 2005; accepted in final form 10 March 2006
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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q/11 type G-protein-coupled receptors (Gq/11/GPCRs), increase prostaglandin (PG) expression. This is not known for the OTRs expressed by central OT neurons. We examined mechanisms underlying OT's effects on supraoptic nucleus (SON) OT and vasopressin (VP) neurons in hypothalamic slices from lactating rats. OT application (10 pM, 10 min) significantly increased firing rates of OT and VP neurons, both of which expressed OTRs. Indomethacin, an inhibitor of PG synthetases, blocked these increases. OTR (but not a V1 receptor) antagonist blocked OT effects without blocking the excitatory effect of PGE2. Tetanus toxin blocked OT effects on fast synaptic inputs and firing activity of SON neurons but not OT-evoked depolarization, suggesting involvement of both pre- and postsynaptic neurons. Indomethacin also blocked the excitatory effects of phenylephrine, another Gq/11/GPCR activating agent but not those of PGE2, a non-Gq/11/GPCR activating agent in the SON. OT or phenylephrine, but not glutamate or KCl, enhanced cyclooxygenase 2 expression at cytosolic loci in SON neurons and nearby astrocytes, as revealed by immunocytochemistry. This OT effect was not blocked by TTX. Western blot analyses showed that OT significantly increased cyclooxygenase 2 but not actin expression. OT promoted the formation of filamentous actin (F-actin) networks at membrane subcortical areas of both OT and VP neurons. Indomethacin blocked enhancement of F-actin networks by OT but not by PGE2. These results indicate that PGs serve as a common mediator of Gq/11/GPCR-activating agents in neuronal function. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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q/11 G protein (Gq/11/GPCRs) initiates intracellular signaling cascades in many neurons, e.g., GnRH neurons (Piva et al. 2004
), melanin-concentrating cells (Griffond and Baker 2002), and lactotrophs and corticotrophs (Suarez et al. 2002). These cascades mainly involve mobilization of phosphatidylinositol 4, 5-bisphosphate (PIP2), subsequent activation of protein kinase C (PKC) by diacylglycerol (DAG), mobilization of intracellular Ca2+ stores, and activation of extracellular signal regulated protein kinase 1/2 (ERK 1/2) (Luttrell 2003). Despite abundant evidence from studies on nonneuronal tissues, our understanding of Gq/11/GPCR signaling pathways in neurons is quite meager.
Oxytocin receptors (OTRs) belong to Gq/11/GPCRs (Di Scala-Guenot and Strosser 1992; Strakova et al. 1998). In uterus, prostaglandins (PGs) are a major product and mediator of OT signaling after activation of OTRs (Molnar et al. 1999; Zlatnik et al. 2000). OT also increases the polymerization of filamentous actin (F-actin) (Gogarten et al. 2001), likely by PGE2 mediation (Martineau et al. 2004). In supraoptic nucleus (SON) neurons, the effect of OT is related to inositol trisphosphate (IP3)-sensitive Ca2+ stores (Ludwig et al. 2002), indicating an activation of Gq/11-associated phospholipase C (PLC). Although PGs have excitatory effects on both OT and vasopressin (VP) neurons (Ibrahim et al. 1999), increased blood OT concentrations (Knigge et al. 2003), and DAG and membrane arachidonic acid (AA) could be converted to PGs, it has not been established that PGs are mediators of OT's actions, especially excitation, in SON neurons. Furthermore, type 1 VP receptors on both OT and VP neurons also belong to Gq/11/GPCRs (Dayanithi et al. 2000), and VP neurons can be activated by OT (Yamashita et al. 1987). If OT's effects on intracellular signals and neuronal excitability represent common responses to Gq/11/GPCR activation, then similar effects of OT should be observed in both OT and VP neurons. The important question, then, is how PGs might mediate OT's feedback effects on OT and VP neurons.
We used several approaches, including whole cell patch-clamp recordings of OT neurons in slices, immunocytochemistry/confocal microscopy for OT, glial fibrillary acidic protein (GFAP), OTRs and cyclooxygenase 2 (Cox-2), and Western blot analyses, to reveal the possible role of PGs in OT actions on OT and VP neurons. The dynamic increase in OT concentration and its autoexcitatory effects during suckling have been firmly established (Neumann et al. 1993). The initial purpose of this study was to explore mechanisms of OT facilitation of burst firing in OT neurons during suckling. Thus the experiments used lactating, not male or virgin female, rats. We verified the presence of OTRs on all SON cells and confirmed a common pathway of PGs in Gq/11/GPCR signaling by comparing the influence of blocking PG synthesis on the excitatory effects of phenylephrine and PGE2. Further, we analyzed the facilitatory effects of OT and PGE2 on the formation of membrane subcortical F-actin networks. We report that PGs, in association with the formation of F-actin networks, are crucial mediators of OT excitatory actions in SON neurons of lactating rats.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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Electrophysiology
Sprague Dawley (Holtzman strain) rats, lactating for 8–13 days, were used for the experiments. Rats were decapitated with a guillotine; brains were quickly removed and placed in ice-cold oxygenated, artificial cerebrospinal fluid (ACSF) for 1 min. The ACSF contained (in mM) 126 NaCl, 2.5 KCl, 1.3 MgSO4, 2.4 CaCl2, 1.3 NaH2PO4, 26 NaHCO3, 10 glucose, and 0.2 ascorbic acid, pH 7.4 adjusted with 3-(N-morpholino) propanesulfonic acid (MOPS, 
2 mM). Osmolality was adjusted to 300 mOsm/kg. The ACSF was filtered (0.22 µm) and maintained with 95% O2-5% CO2 gas mixture. Hypothalami were dissected from the brain and cut coronally at 300 µm on a Vibratome. After preincubation at room temperature (RT, 21-23°C) for 
1 h, whole cell patch-clamp electrophysiological recordings of membrane potential (Em) and action potentials were made at 35°C. Patch pipette filling solution contained (in mM) 145 K-gluconate, 10 KCl, 1 MgCl2, 10 HEPES, 1 EGTA, 0.01 CaCl2, 2 Mg-ATP, and 0.5 Na2-GTP, pH 7.3, adjusted with KOH. For immunocytochemical identification of recorded neurons, 0.05% Lucifer yellow (LY) was added to the pipette solution. Patch electrodes were guided onto SON cells under visual observation through an upright microscope (Leica DM LFSA) equipped with water-immersion objectives, IR/DIC and filters for fluorescence microscopy. Whole cell recordings were obtained from the somata of SON magnocellular neurons during perfusion of ACSF via a gravity-fed perifusion system at a rate of 1.2–1.5 ml/min. An Axoclamp 2B amplifier was used for collecting electrical signals that were filtered and sampled at 5 kHz by Clampex 9 software through a 1320 AD/DA converter (Axon Instruments). Series resistance compensation was between 60 and 80%. Data were stored in a PC computer for off-line analysis. Measured liquid junction potentials of –8 to –11 mV (potential of pipette solution with respect to the bath) were uncorrected in the results, because the variation of pipette tips brought 2- to 3-mV difference among individual recordings and made accurate estimates of compensation difficult.
Immunocytochemistry
The methods for identification of recorded neurons were modified from previous publication (Smithson and Hatton 1990). After recording with electrodes containing LY in the pipette solution, slices were fixed in 4% paraformaldehyde at 4°C overnight and treated with 0.3% Triton X-100 for 30 min. Slices were then incubated with a goat polyclonal antibody against either OT- or VP- neurophysin (OT-NP or VP-NP at 1:250 dilution) along with a monoclonal antibody against other NP, i.e., either VP-NP (PS41) or OT-NP (PS38) at 1:1,000 for 4 h at RT before the secondary antibody was applied. Primary antibodies: goat polyclonal antibody against OTR or Cox-2 (1:200 dilution), rabbit polyclonal antibody against GFAP (1:200) and monoclonal antibody against OT-NP or VP-NP (PS38 or PS41, 1:1,000) were applied. Secondary antibodies: donkey anti-goat antibody (Alexa Fluor 647 labeled, 1:1,000), donkey anti-rabbit antibody (Alexa Fluor 488 labeled, 1:1,000) and donkey anti-mouse antibody (Alexa Fluor 555 labeled, 1:1,000) were applied for 1.5 h to label these primary antibodies. To identify F-actin, Alex Fluor 488-conjugated phalloidin (instead of Alexa Fluor 488-labeled donkey anti-rabbit antibody) was applied for 30 min, together with other secondary antibodies. Finally, Hoechst (bisbenzimide, 1:1,000) was applied for 30 min to label the nuclei. Sections were sealed on glass slides with Vectashield to avoid bleaching. Images of neurons between 10 and 40 µm from the surface of the slices were examined with a laser scanning confocal microscope (Leica TCP SP2) in sequential and Z-series scanning modes. Overlap in the different merged images was taken as evidence of co-localization.
Western blot analysis
SONs in hypothalamic slices were punched out with a micro-circular cutter, and then trimmed so that each punch contained only the SON (1 mg/SON wet weight). After preincubation at RT for 1 h, the punches were treated with OT and then transferred into a cold lysis buffer for dissociation of tissues with sonication. The lysates were centrifuged at 13,000 rpm for 10 min at 4°C. Protein concentration in the supernatant was assayed using Bio-Rad Protein Assay (Bio-Rad Laboratories, Hercules, CA). Protein samples (40 µg each) were separated on 10% sodium dodecyl sulfate polyacrylamide gels. Protein was transferred onto a nitrocellulose membrane at 4°C. Membranes were blocked with 5% milk solids for 1 h at RT, then incubated with polyclonal goat Cox-2 antibody (1:200) or polyclonal rabbit actin antibody (1:500), and re-probed for 3 h at RT after stripping off Cox-2 antibody. Cox-2 and actin were visualized using horseradish peroxidase-conjugated secondary antibodies and an enhanced chemiluminencence system. Positive bands for Cox-2 were confirmed by Cox-2 positive control.
Reagents
All chemicals were from Sigma (St. Louis, MO) except otherwise noted. Goat polyclonal antibodies against OT-NP, N-terminals of OTRs and Cox-2, and Cox-2 positive control (RAW 264.7+LPS/PMA cell lysates) were from Santa Cruz Biotechnology (Santa Cruz, CA). Monoclonal antibodies against OT-NP (PS38) and VP-NP (PS41) were kindly provided by Dr. H. Gainer (National Institutes of Health). Rabbit polyclonal antibody against GFAP was from DAKO (Denmark). Alexa Fluor 488 (555, or 647)-labeled secondary antibodies and Alex Fluor 488 conjugated phalloidin were from Molecular Probes (Eugene, OR). Reagents for Western blots were from Amersham Biosciences (Piscataway, NJ) and Bio-Rad Laboratories (Hercules, CA) and the HRP conjugated goat IgG antibody was from (BETHYL, Montgomery, TX). Vectashield was purchased from Vector Laboratories (Burlingame, CA).
Data collection and analysis
Neurons that fired continuously and showed sustained outward rectification (SOR) in response to 11 steps (5 mV/step) of hyperpolarizing pulses (each lasting 1,200 ms) from –40 mV (Stern and Armstrong 1995) were taken as putative OT neurons. The SOR assessment was always performed at the very beginning of whole cell recording, to ensure that no significant dialysis had occurred (Wang and Hatton 2004). Putative VP neurons were those that fired phasically and showed clear plateau potentials. In our analysis of phasically firing neurons, we adopted the criteria used by Leng and colleagues (Brown et al. 1998). Neurons that fired irregularly were only identified subsequently by immunocytochemistry, and silent neurons were not analyzed in this study. In evaluating the relative intensity of different immunostaining, the luminosity of neurons in a specific channel in each section was assayed with Photoshop (6.0). A value for each section (slice) was obtained by averaging values from 6 to12 neurons or as otherwise indicated in RESULTS. In the same series of experiments, depth of focus, photo multiplier tube (PMT) intensity (350–600 V), offset (–1–4%), pinhole size (1 airy unit corresponding to an optical slice thickness of <300 nm), magnification (x63 objective lens) and zoom (3–5 times) were kept at the same value. Differences >20% in fluorescence intensity for various treatments were considered to be different. Merging of different channels was confirmed by Z-series scanning. The intensity of Cox-2 protein staining in Western blots was determined by multiplying average luminosity by the pixel size of corresponding bands with Photoshop software. The criteria for evaluating F-actin structures were as follows. Confocal images of three horizontal and three vertical sections, spaced equidistant from one another in one neuron, were measured with Leica LCS Lite software. Neurons that had intact membrane subcortical F-actin networks (or F-actin ring) were required to show two peaks in gray scale, in each intersection, that were at the periphery of each neuron. This feature had to be consistent through sections in Z-series scanning. Neurons exhibiting only one peak or no peak in any of the six measurements were considered to lack intact membrane subcortical F-actin networks. ANOVA, paired t-test or Wilcoxon rank test and 
2 tests were used for statistical analyses by SigmaStat (SPSS), and P < 0.05 was considered significant. All measures were expressed as means ± SE except as otherwise indicated in RESULTS.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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Excitatory effects of OT via PG synthesis
Experimental manipulations began only after the OT neuron was judged to have become electrophysiologically stable in whole cell configuration. OT (10 pM, 10 min) was then bath applied (Fig. 1A). Ten of 11 OT neurons tested showed increased firing rates >20% at the end of 10 min (Fig. 1A1), a small but significant increase (Fig. 1A, 2 and 3; P < 0.05, n = 11). Meanwhile, there was a relatively large increase in membrane potential (Em) depolarization (P < 0.01). This result is in agreement with previous work (Yamashita et al. 1987).
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). If OT acts via PGs, blocking PG synthetases should occlude OT excitatory effects. Therefore we investigated the effect of OT during a blockade of PG production. Bath application of 1 µM indomethacin (an inhibitor of cyclooxygenases, enzymes for PG synthesis) for 10 min significantly reduced the basal firing rate (Fig. 1B, P < 0.05, n = 8) and hyperpolarized the Em in most of the cells (5/8). Preincubation of the slices with indomethacin blocked excitatory responses and the OT-induced Em depolarization (Fig. 1B, 2 and 3), suggesting that OT excitatory effects are mediated by PGs. In parallel with the effect on OT neurons, the same dose of OT, but not VP, caused significant Em depolarization and excitation in VP neurons (Fig. 2A, P < 0.01, n = 16). In the presence of indomethacin, excitatory responses and the Em depolarization to OT were blocked (Fig. 2B, P > 0.05, n = 7), suggesting that PGs also mediate the excitatory effects of OT on VP neurons. In comparison, the significant (P < 0.05) effects of OT on OT neuronal firing rate occurred earlier than in VP neurons (5 vs. 9 min in minute-by-minute analysis). However, OT caused a more profound increase in peak firing rate of VP neurons (185% in OT vs. 340% in VP neurons of control). This is likely associated with the phasic bursting features of VP neurons.
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OTRs mediate the excitatory effects of OT
The excitatory effects of OT on both OT and VP neurons raised an essential question: what receptors mediate OT's effects. It is known that OT can activate both OTRs and V1 receptors in SON neurons. Here we further examined the specificity of OT actions. In the presence of a selective OTR antagonist, [
-mercapto-, -cyclopentamethylene-propionyl1, O-Me-Tyr2, Orn8]-oxytocin, the excitatory effects of OT on both OT (n = 6) and VP neurons (n = 6) were blocked (Fig. 3A). The selective V1 receptor antagonist, [deamino-Pen1, O-Me-Tyr2, Arg8]-vasopressin, however, did not significantly influence the excitatory effects of OT on either OT (n = 6) or VP (n = 8) neurons (Fig. 3B). The excitatory effects of OT on VP neurons were also manifested in reduced inter-burst intervals (from 399 ± 146 to 163 ± 44 s, 0.05 < P < 0.1), increased burst duration (from 174 ± 68 to 1041 ± 332 s, P < 0.05) and increased intra-burst firing rates (from 2.9 ± 0.9 to 4.5 ± 0.8 Hz, 0.05 < P < 0.1). Together with previous findings, these results indicate that the excitatory effect of OT is achieved via activation of OTRs but not via V1 receptors. Moreover, blocking OTRs did not block PGE2 excitatory effects on either cell type, as shown by the two examples in Fig. 3D.
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The activity of OT neurons heavily depends on afferent inputs (Wang et al. 2006). Glutamatergic and GABAergic inputs are the main sources of direct synaptic contacts with OT neurons (Armstrong and Stern 1997; Theodosis et al. 1995). Here we used tetanus toxin (10 nM) pretreatment of slices for 1–1.5 h, then observed the effect of OT on excitatory/inhibitory postsynaptic currents (EPSCs/IPSCs) and firing activity. Without this treatment, OT significantly reduced the EPSCs and IPSCs (data not shown), consistent with previous reports at higher doses of OT (Brussaard et al. 1996; Kombian et al. 1997). After tetanus toxin treatment, EPSCs precipitously declined in size and frequency, and IPSCs actually disappeared. No marked changes in slow membrane currents were observed in response to OT (Fig. 5A, 1 and 2). Pretreatment with Tetanus toxin significantly hyperpolarized the Em (–55.3 ± 1.2 vs. –52.3 ± 1.0 mV in control, P < 0.05, n = 8 for each group). OT still had a depolarizing effect (–56.6 ± 1.2 vs. –54.1 ± 1.4 mV, P < 0.05), on both OT (n = 4) and VP neurons (n = 4), whereas firing activity was unchanged for the group as a whole (Fig. 5B1). However, in one VP neuron with relatively depolarized membrane potential, OT did increase its firing rate (Fig. 5B2). These results indicate that OT does have postsynaptic effects, but its excitatory effect heavily depends on synaptic vesicle release and the basal Em level. Because OT did not change the membrane conductance, and the excitatory action was relatively slow compared with the actions of channel-coupled receptors of neurotransmitters, we extended our study by looking for PG-associated intracellular signaling rather than OT effect on ionic channel activity (although OT did decrease voltage-gated Na+ current in slices with longer application, data not shown here).
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q/11/GPCR-activating agents
The excitatory effects of OT on both OT and VP neurons and their elimination by blockade of PG synthesis highlight the possibility that PGs are a common mediator in actions of Gq/11/GPCR-activating agents. However, the possibility remains that indomethacin worked separately from Gq/11/GPCR signaling. Therefore further experiments were performed on OT and VP neurons based on the common responses to OT observed in the two types of SON neurons. First, the influence of indomethacin on the effects of phenylephrine, another Gq/11/GPCR-activating agent, was observed. As is commonly observed, phenylephrine (10 µM, 5 min) caused significant excitation in five of five SON neurons (Fig. 6A). The presence of indomethacin (1 µM, 10 min before) blocked the excitatory effect of phenylephrine (Fig. 6A) in four of four OT neurons and three of three VP neurons. This result strongly suggests that the mediation of PGs in excitatory responses caused by OT is a common effect of activating Gq/11/GPCRs. To eliminate the possibility that the ineffectiveness of OT or phenylephrine in the presence of indomethacin was the result of a nonspecific inhibitory effect of indomethacin, we injected positive current to re-excite neurons by direct depolarization of Em (by 6–12 mV) at 10 min after indomethacin application. In all 6 neurons tested (3 OT and 3 VP neurons), OT still failed to significantly change the excitability or Em levels of SON neurons (Fig. 6B), suggesting that the blockade of OT and phenylephrine actions was not due to nonspecific inhibitory effects of indomethacin. To further strengthen the preceding conclusion and confirm a specific mediation by PGs in Gq/11/GPCR signaling, we observed the effect of PGE2, a non-Gq/11/GPCR-activating agent for SON neurons. In four of four OT neurons and three of three VP neurons, the presence of indomethacin did not block the excitatory effect of PGE2 (Fig. 6C), confirming a specific effect for indomethacin on PG synthesis but not on PGE2's excitatory action.
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Further experiments confirmed that OT increases the expression of Cox-2 in the SON. Twelve paired hemi-slices from six rats were randomly assigned to control and OT treated groups. These 12 hemi-slices were subsequently subjected to immunocytochemical analyses for OT, VP, Cox-2, and, in three cases, GFAP. They were immuno-identified as OT-Cox-2 (3 pairs with GFAP) and VP-Cox-2, respectively. Before application of OT, 73.5% of the SON neurons (n = 54) showed Cox-2-positive staining, which was highly concentrated in nuclear areas, and only 20% showed visible cytosolic Cox-2 (i.e., >20% above background levels). Application of 10 pM OT for 5 min significantly (6/6, P < 0.05) increased the total number of Cox-2-positive OT neurons (94.5%, n = 64) and GFAP-positive cells (3/3; Fig. 7A). The number of OT neurons that were Cox-2 positive in cytosolic areas increased significantly (to 85% in OT treated groups, P < 0.01 by 2 test). In particular, the relative intensity of Cox-2 staining in cytosolic areas was increased significantly (Fig. 7C1), suggesting that OT activates PG synthesis in both OT neurons and astrocytes. Cox-2 activation by OT in OT neurons was accompanied by Cox-2 activation in VP neurons (Fig. 7B). Similar to OT neurons, under control conditions, Cox-2-positive immunoreactivity was concentrated in nuclear areas. Weak cytosolic staining in 39% of the VP neurons was observed in six hemi-slices. OT treatment did not significantly change the ratio of Cox-2 positive to negative VP neurons (before 76.7%, n = 67; after 80.8%, n = 56). However, the intensity of Cox-2 staining that was particularly dramatic in cytosolic areas increased significantly in all six slices (Fig. 7C2). In the presence of indomethacin (1 µM for 10 min), no cytosolic Cox-2 was detected; however, nuclear Cox-2 expression was not influenced markedly. Addition of OT (10 pM for 5 min) slightly increased the expression of cytosolic Cox-2 expression (12.5% of 16 neurons), whereas there were no obvious changes in nuclear Cox-2 expression, indicating the function of indomethacin at Cox-2 levels. Finally, Western blot analysis demonstrated that 5 min of OT application significantly (P < 0.05, n = 4) increased Cox-2 protein expression in the SON, whereas the total amount of actin (re-probed after stripping Cox-2 antibodies) was not changed significantly (Fig. 7D).
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q/11/GPCR activation but simply to increased firing activity. To remove this doubt, we performed the following experiments in two rats with four hemi-slices in each group. With action potentials blocked by TTX (1 µM for 30 min), the basal cytosolic Cox-2 expression in SON neurons (75% of 16 OT neurons and non-OT neurons) was significantly higher than in TTX-free normal medium, whereas nuclear Cox-2 expression was obviously reduced (37.5% of the cells). Addition of OT in the presence of TTX actually activated all of the neurons, including nuclear sites, and astrocytes (Fig. 8A). Although this result supports a direct postsynaptic action, it raises the suspicion that it may be the action potentials that induced Cox-2 expression. In support of a specific action for OT, we further tested the effect of 0.1 mM glutamate and 12 mM KCl on Cox-2 expression, which are well known for their excitatory effects on SON neurons. Treatment of four hemi-slices with glutamate did not significantly influence (actually appeared to reduce) the expression of Cox-2 at cytosolic sites in 16 SON neurons (6.3%) and intermingled GFAP-positive components (Fig. 8B). Similarly, high K+ solution for 5 min did not increase the number of neurons that were cytosolic Cox-2 positive (12.5%) nor the number of astrocytes (Fig. 8C). By contrast, 10 µM phenylephrine significantly increased the number of cytosolic Cox-2-positive OT neurons, whereas no strong activation appeared at GFAP-positive elements (Fig. 8D). It was noted that the ratio of Cox-2-positive cells (43.8% of 16 OT neurons in 4 hemi-slices) in the phenylephrine-treated group was obviously lower than that resulting from OT treatment. These results suggest that action potentials or their associated synaptic transmission exerted an inhibitory rather than an excitatory effect on the induction of Cox-2 expression by OT. Alternatively, it is very likely for PGs to mediate effects of OTRs and other Gq/11/GPCR at least in SON neurons.
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Potential involvement of PGs in OT actions was further examined with the expression of F-actin, a target of PG actions in peripheral organs. In six paired hemi-slices from the rats used in the experiments described in the preceding text, OT-NP and F-actin were immunostained. In the control condition, weak or no expression of intact F-actin networks was seen in both OT (n = 36) and non-OT (n = 36) neurons at membrane subcortical areas (Fig. 9A1). Bath application of OT for 5 min significantly increased the formation of F-actin networks at membrane subcortical areas in OT (35/36) and non-OT (33/36) neurons (Fig. 9A2). Comparing average luminosities of F-actin at corresponding subcortical areas in OT neurons of different slices (n = 6), a significant increase was observed after OT treatment (control, 94.2 ± 16.4; OT, 116.2 ± 10.7, P < 0.01). Pretreatment of slices with indomethacin did not significantly influence F-actin ring-like structure in OT or non-OT neurons compared with control. The presence of indomethacin also blocked OT (5.6%, n = 2/36)- but not PGE2 (91.7%, n = 33/36)-induced formation of F-actin networks (Fig. 9B, 1 and 2). Similar to the OT effects, phenylephrine increased the number of F-actin networks at membrane subcortical areas; however, the ratio was lower (62.5% of 16 neurons) than for OT. This result suggests that OT increased F-actin networks at membrane subcortical areas via production of PGs. Because Western blot analysis revealed no significant changes in actin expression, it seems that the changes in F-actin imaging mainly reflect structural changes and spatiotemporal redistribution of actin molecules, rather than an alteration in synthesis.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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q/11/GPCR signaling. This mediation likely includes PG-induced reorganization of actin cytoskeletal elements.
OT has been reported to have several actions in the SON via OTRs. These include electrical excitation (Freund-Mercier and Richard 1984; Inenaga and Yamashita 1986; Yamashita et al. 1987), reduction of GABAergic (Brussaard et al. 1996) or glutamatergic synaptic inputs (Hirasawa et al. 2001; Kombian et al. 1997), autocontrol of OT release (Chevaleyre et al. 2000; de Kock et al. 2003), and mobilization of intracellular Ca2+ levels (Lambert et al. 1994). Different from most of the literature available in this field suggesting that OT excites only OT neurons, our results showed excitatory effects of OT on both OT and VP neurons. This discrepancy is apparently due to several differences in experimental conditions, observation periods, etc. Without much data to support such a notion, most of the literature available in this field also suggests or infers that OTRs are located exclusively on OT-secreting cells and that OT excites only OT neurons. Analysis of experimental conditions used earlier, along with new data pertinent to these issues, yields a somewhat different picture. First, the times of OT application differ. Most previous experiments observed effects of OT for shorter times (<5 min), so would have missed OT effects on VP neurons which occurred at times >5 min. A second crucial difference is OT dose. We used a relatively low level (10 pM OT) and most others used 1 nM to 1 µM of OT. Because OTRs are composed of different subtypes (Gimpl and Fahrenholz 2001), our low dose may reflect the effect of OT on a high-affinity, low-volume type. Third, different routes of OT application may yield different results. OT or VP injection into the third ventricle had no effect on VP neurons (Freund-Mercier and Richard 1984), but in slices, VP can influence synaptic activity of OT neurons (Hirasawa et al. 2003). This could explain why an in vivo study (Freund-Mercier and Richard 1984) showed no OT effects on VP neuronal firing, whereas the present clearly observed these. Fourth, in patch-clamp recordings (from cells mostly at the surface of the slice), SON neurons, likely surrounded by very low concentrations of endogenous OT due to medium perifusion, had higher accessibility and sensitivity to exogenous OT than would neurons recorded at sites deeper in the slice in either extracellular or sharp electrode recordings. Patch recordings also avoid negative influences of spatial averaging of extracellular currents or leakage around sharp electrodes. The excitatory effects elicited by low OT doses are in agreement with basal OT levels measured in the CSF: <10 pM in direct measurement in humans (Altemus et al. 2004) and rats (Devarajana and Rusak 2004) and supported by the excitatory actions of exogenous OT at 50 pM (Kuriyama et al. 1993) in extracellular recordings. A fifth factor may be the care taken to avoid desensitization by thoroughly cleaning perfusion system between tests. Because SON neurons are highly sensitive to OT, any residual OT from previous tests could reduce the sensitivity of VP neurons. Actually, in Yamashita's study (Yamashita et al. 1987), OT did excite a small percentage of phasically firing SON neurons.
The most powerful argument in this regard is our immunocytochemical evidence (antibody targeting the OTR N-terminus) showing OTR expression on both OT and VP cell types as well as on astrocytes of the SON. This provides a strong basis for direct OT activation of the various SON cell types.
In the periphery, a dramatic effect of OT is to increase production of PGs (Jeng et al. 2000). That OT also increases PG synthesis in SON neurons and their associated astrocytes confirms previous reports (Mouihate et al. 2002; Ojeda et al. 2000). This OT-induced increase in PG production can be achieved through activating phospholipase A2 and cyclooxygenases via activating ERK 1/2 (Zlatnik et al. 2000) and by raising intracellular Ca2+ (Jeng et al. 2000). Activation of OTRs increases intracellular Ca2+ (Ludwig et al. 2002), and the activity of PKC (Koksma et al. 2003). Increases in intracellular Ca2+ in SON neurons by OT (Di Scala-Guenot et al. 1994; Lambert et al. 1994) can cause membrane translocation of phospholipase A2, which releases AA from plasma membranes. The breakdown of PIP2 by PLC also increases substrates for PG production by releasing DAG. Our recent work has confirmed OT activation of ERK 1/2 in SON neurons (Hatton and Wang 2005). Activation of ERK 1/2 will further phosphorylate and activate phospholipase A2 (Chakraborti 2003) and cyclooxygenases (Jeng et al. 2000), enhancing PG synthesis.
The increased Cox-2 expression and potentially increased production of PGs are partially responsible for OT's autoexcitatory effects and represent a common effect of activating Gq/11/GPCR. This proposal is supported by the observation that blocking PG synthesis also blocked OT- and phenylephrine-, but not PGE2- evoked excitation and F-actin network formation in both OT and VP neurons. Because both OT and phenylephrine function via activation of Gq/11/GPCRs, whereas PGE2 does not, the blocking effects of indomethacin can be explained as the specific blockade of PG synthesis, a key link in Gq/11/GPCR signaling. To reinforce this, neither glutamate nor KCl increased Cox-2 expression, although they are well known to excite neurons. It is unlikely that the ineffectiveness of OT in the presence of indomethacin was derived from inhibitory effects of indomethacin itself. If this were the case, then PGE2 application would not have had excitatory effects, and OT would have continued to excite SON neurons after Em was depolarized to remove potential nonspecific inhibition. Neither was observed.
The actions of OT involve both pre- and postsynaptic neurons. Tetanus toxin blocked synaptic vesicle release but not OT depolarizing effects. TTX blockade of spontaneous postsynaptic Na+ currents did not preclude OT-induced enhanced Cox-2 expression. Because the effects of OT are not necessarily related to the firing activity at somata (Ludwig et al. 2002), that there was no effect on membrane conductance and little change in firing activity after tetanus toxin treatment, may support the hypothesis that the electrical activity of SON neurons under OT stimulation heavily depends on presynaptic inputs and astrocyte-originated transmission. Direct actions of OT on SON neurons are closely associated with the intracellular metabolic and slow responses, e.g., PG production, and burst generation. Together with previous reports (Brussaard et al. 1996; Chevaleyre et al. 2000; de Kock et al. 2003; Hirasawa et al. 2001; Kombian et al. 1997; Lambert et al. 1994), we conclude that both pre- and postsynaptic neurons mediate OT actions.
The mechanisms underlying PG excitatory effects in OT actions may involve several signaling pathways. Studies have shown that PG excitatory effects were partially mediated presynaptically through an inhibition of GABA release via EP3 receptors (Shibuya et al. 2000). PGs had a postsynaptic excitatory action on SON neurons mediated by activation of cationic currents, also in dissociated SON neurons, and the reversal potential of these currents was –35.5 ± 0.9 mV (Sutarmo Setiadji et al. 1998) via EP4 receptors. PGs increase hyperpolarization activated inward current (Ingram and Williams 1996) and reduce K+ conductance (Nicol et al. 1997). These effects are consistent with known mechanisms underlying OT-mediated excitatory responses in neurons. For example, in brain stem motoneurons or in spinal cord neurons, OT increases neuronal excitability via actions that result in opening nonselective cation channels or that close K+ channels (Raggenbass 2001). Thus it is likely that OT acts at both SON neurons and astrocytes leading to increased release of PGs, which together with other neurotransmitters strengthen OT actions by modulating the activity of both pre- and postsynaptic neurons.
It was previously unknown whether PGs also function through modulation of actin dynamics. The present study provides the first evidence that OT increases the formation of F-actin in neurons through PGs, raising the possibility that dynamic F-actin plays a key role generally in the functions of OT and PGs. Blocking PG synthesis blocked the formation of subcortical F-actin networks, while OT's excitatory effect was also reduced significantly. The mediation of PGs in OT's promoting actin polymerization is also clear: blocking PG synthesis did not block PGE2-evoked actin dynamics. In fact, our study did find that destruction of actin cytoskeleton by cytochalasin B caused a prolonged inhibition of the firing activity, directly or after a short excitation. The presence of cytochalasin B for 15 min was enough to block the excitatory effects of OT; applying OT for >30 min often caused disruption of actin cytoskeleton, which is accompanied by dramatic spike frequency reduction (unpublished data). Thus although we could not completely exclude the possibility that F-actin is merely a co-factor in OT-PG-evoked excitation and not a crucial link in OT-PG signaling cascades, the probability is high that dynamic F-actin reorganization is, indeed, a crucial link in OT-PG-mediated excitation.
Although we established presence of the preceding signaling cascade, we cannot exclude the possibility that OT and PGs could work in balance or parallel. Indomethacin did not completely block the effect of OT. This could be due to an incomplete effect of indomethacin in blocking Cox-2 activation or because of the effect of divergent pathways downstream of Gq/11/GPCR, other than PGs (Ca2+, PKC, etc). It is also true that activation of OTRs, 1-adrenoceptors and likely many other neurotransmitter receptors can increase PG synthesis. We conclude that PG is one of the important pathways mediating Gq/11 type G-Protein actions, but not only one.
Implications of our current findings are broad. Excitation of both OT and VP neurons by OT may reflect the functional harmony of the two peptide types in fulfilling the milk-ejection reflex. Facing an increased loss of extracellular fluids during suckling of the young, VP initiates a water-conservation process that maintains plasma volume, allowing prolonged suckling (Grindstaff and Cunningham 2001). The excitation of OT neurons through PGs may be a key link for OT-evoked bursts (Hatton and Wang 2005). This signal transduction cascade in SON neurons may serve as a general model for many, if not all Gq/11/GPCRs in mammalian neurons. Nevertheless, different neurons have their own subtypes of Gq/11/GPCRs, and the same PGs may cause different effects in neurons with different PG receptor subtypes. However, that activating Gq/11/GPCRs increased synthesis of PGs in response to many neurotransmitters or neuromodulators does support the existence of such common intracellular signaling pathways, e.g., metabotropic glutamate receptors of the mGlu1 and mGlu5 subtypes (Pellegrini-Giampietro 2003) and angiotensin type 1B receptors (Suarez et al. 2002). Moreover, PGE2 can extensively activate neurons, as indicated by enhanced expression of c-fos mRNA (Lacroix et al. 1996). PGs, as diffusible, membrane-permeable neuromodulators may be particularly potent in causing secondary effects. The delayed excitatory responses of VP neurons to OT, and the irrelevance of V1 receptor in OT actions may partially reflect this point. Recognizing this secondary effect of PG production (Lopez Bernal et al. 1995) and possibly other secondary cytokines (e.g., cannabinoids, opioids, etc.) in OT actions may reconcile the existing controversial reports about involvement of different GPCRs in actions of individual neuropeptides.
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Address for reprint requests and other correspondence: Y.-F. Wang, Dept. of Cell Biology and Neuroscience, University of California, Riverside, CA 92521 (E-mail: yufengw{at}ucr.edu)
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