AJP - Regu AJP: Endocrinology and Metabolism
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Regul Integr Comp Physiol 280: R313-R322, 2001;
0363-6119/01 $5.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (5)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Morsette, D. J.
Right arrow Articles by Sladek, C. D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Morsette, D. J.
Right arrow Articles by Sladek, C. D.
Vol. 280, Issue 2, R313-R322, February 2001

Role of non-NMDA receptors in vasopressin and oxytocin release from rat hypothalamo-neurohypophysial explants

Delmore J. Morsette, Hanna Sidorowicz, and Celia D. Sladek

Department of Physiology and Biophysics, Finch University of Health Sciences The Chicago Medical School, North Chicago, Illinois 60064


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Glutamate is recognized as a prominent excitatory transmitter in the supraoptic nucleus (SON) and is involved in transmission of osmoregulatory information from the osmoreceptors to the vasopressin (VP) and oxytocin (OT) neurons. Explants of the hypothalamo-neurohypophysial system were utilized to characterize the roles of the non-N-methyl-D-aspartate (NMDA) glutamate receptor subtypes (non-NMDA-Rs), kainic acid receptors (KA-Rs), and aminopropionic acid receptors (AMPA-Rs) and to evaluate the interdependence of NMDA-Rs and non-NMDA-Rs in eliciting hormone release. Although both KA and AMPA increased hormone release, a specific agonist of the KA-Rs, SYM-2081, was not effective. This combined with the finding that cyclothiazide, an agent that inhibits the desensitization of AMPA-Rs, increased the VP response to both KA and AMPA indicates that the increase in hormone release induced by the non-NMDA agonists is mediated via AMPA-Rs, rather than KA-Rs. Inhibition of osmotically stimulated VP and OT release by a specific AMPA-R antagonist indicated that AMPA-Rs are essential for mediating osmotically stimulated hormone release. NMDA-stimulated VP but not OT release was prevented by blockade of non-NMDA-Rs, but AMPA-stimulated VP/OT release was not prevented by NMDA-R blockade.

glutamate; kainate; aminopropionic acid; osmolality; supraoptic nucleus; N-methyl-D-aspartate


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

GLUTAMATE IS AN IMPORTANT excitatory neurotransmitter in the hypothalamus, as demonstrated by extensive morphological and electrophysiological studies (18, 19, 44). Glutamate interacts with two classes of ionotropic receptors, N-methyl-D-aspartate (NMDA-R) and non-NMDA (nonNMDA-Rs). Both groups are expressed in the supraoptic nucleus (SON), as demonstrated by immunocytochemistry, in situ hybridization, and electrophysiological techniques (1, 2, 11, 12, 21, 22, 28). Glutamate is the primary excitatory agent transmitting osmotic information from the organum vasculosum of the lamina terminalis (OVLT) to the VP and OT neurons (29). Excitatory postsynaptic potentials can be recorded in SON upon stimulation of the osmoreceptive region of the OVLT. The pattern of this activation contains two components: one that can be blocked by a non-NMDA-R antagonist and a second that can be blocked by a NMDA-R specific antagonist (48). Because both NMDA and non-NMDA-R antagonists also block osmotically stimulated VP release from explants of the hypothalamo-neurohypophysial systyem (HNS; 35, 40), these data suggest an important role for glutamate in mediating osmotic activation of SON neurons. However, the respective roles of the NMDA and non-NMDA-R subtypes and the interaction between these receptors and the NMDA-Rs on hormone release remain to be elucidated.

The non-NMDA-Rs consist of two groups, the kainic acid receptors (KA-Rs) and the aminopropionic acid receptors (AMPA-Rs). Expression of all the different subunits of AMPA-Rs, as well as several KA-Rs, glutamate receptor (GluR)5,6,7, and KA-2, have been demonstrated morphologically in the SON and hypothalamus (10, 17, 28, 31, 43). Furthermore, electrophysiological results support non-NMDA-R involvement in the activation of VP and OT neurons (22, 39). In the evaluation of the relative contribution of KA-R and AMPA-Rs on hormone release, two issues were considered. The first was the relative lack of selectivity of the agonists. Although AMPA-Rs and KA-Rs are classified according to pharmacological sensitivity to these agonists, KA activates both classes of receptors with relatively poor selectivity (13). Therefore, a more specific KA-R agonist was also used. The second issue was the impact of receptor desensitization on hormone responses. AMPA-Rs desensitize rapidly upon agonist application (24, 25). Therefore, cyclothiazide, a specific blocker of AMPA-R desensitization, was utilized to study AMPA-R involvement in VP and OT release (25).

An important role for NMDA-Rs in initiating hormone release is supported by numerous scientific results including the finding that SON is one of the most densely labeled brain regions for the NMDA-R subunit, NR1 (32). The NMDA-Rs are unique because they are both ligand gated and voltage sensitive. Magnesium blocks the ion channel, and this inhibition is removed upon membrane depolarization (23). In response to glutamate, the non-NMDA-Rs depolarize the membrane, allowing for magnesium removal and activation of NMDA-Rs. This voltage-sensitive characteristic of the NMDA-R has been implicated in inducing bursting activity in VP neurons (14, 21, 22). Because bursting activity facilitates sustained VP release, coactivation of the non-NMDA-Rs and NMDA-Rs may be a requirement for detectable hormone release. Therefore, a codependency between these GluRs may exist in SON to allow sustained hormone release. To test the hypothesis that simultaneous activation of non-NMDA and NMDA-Rs is required for glutamate to stimulate VP and OT release, the effect of NMDA was evaluated in the presence of non-NMDA-R blockade and the effects of AMPA and KA were evaluated in the presence of NMDA-R blockade.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Preparation and perifusion of HNS. HNS explants were obtained from decapitated male Sprague-Dawley rats (125-150 g). The explants included the OT and VP magnocellular neurons of the SON with their axonal projections extending through the median eminence and terminating in the neural lobe. The explants also included the OVLT, the suprachiasmatic nucleus, and the arcuate nucleus. To prepare the explant, the brain was removed from the skull using a caudal approach to maintain the pituitary stalk intact. Under a dissecting microscope, the anterior pituitary was removed along with the dura mater and arachnoid layers. A block of tissue was removed by cutting rostral to the optic tracts, lateral to the median eminence, and caudal to the pituitary stalk. The explant is ~1-2 mm thick and at the rostral end is ~5 mm wide at its widest point. Animal care and experimental protocols were approved by the Institutional Animal Care and Use Committee of the Chicago Medical School and correspond to guidelines of the National Institutes of Health, Bethesda, MD.

HNS explants were perifused at 37°C with oxygenated culture medium. The explants were placed in closed chambers (0.5 ml) and perifused at a rate of 1.8-3.0 ml/h. The medium used was F-12 nutrient mixture (GIBCO or Sigma). The medium was fortified with 20% fetal bovine serum, 1 mg/ml glucose, 50 U/ml penicillin, 50 µg/µl streptomycin, and 0.1 µM bacitracin. Bacitracin was utilized to prevent degradation of the OT and VP in the collected media. Six explants were perifused simultaneously during each experiment. The medium from each chamber was collected at 5°C in a refrigerated fraction collector and stored at -20°C until the OT or VP content was determined by radioimmunoassay. In some experiments, the explants were frozen in liquid nitrogen at the end of the perifusion and stored at -80°C until extracted for RNA.

Experimental procedures. The explants were perifused for 4 h before application of any pharmacological agent or change in osmolality to allow the explant to equilibrate and to establish a basal rate of hormone release. Subsequent hormone release was normalized to the basal release for that explant at the end of the equilibration period and is expressed as percentage of this basal value. When exposed to increased osmolality, the osmolality of the perifusate was increased over a 6-h period by 30 mosmol/kgH2O with NaCl.

Explants were exposed to the following GluR agonists: KA (10 µM-1 mM; Sigma); AMPA (10-500 µM; Tocris); (2S,4R)-4-methylglutamate (SYM-2081; 0.5 µM; Tocris), a KA-R specific agonist (9); and NMDA (50 µM, Tocris). The following GluR antagonists were used: CNQX, an antagonist of KA-Rs and AMPA-Rs (10 µM, Tocris); AP5, an antagonist of NMDA-Rs (100 µM); and SYM-2206 (±-4-(4-aminophenyl)-1,2-dihydro-1-methyl-2-propylcarbamoyl-6,7-methylenedioxyphthalazine, 6 µM, Tocris), a specific AMPA-R antagonist (26). Cyclothiazide (CYC, 100 µM, RBI/Sigma) was also used to inhibit desensitization of AMPA-Rs (25). When the drugs were water insoluble, an appropriate solvent was used, and the control explants were exposed to the same concentration of solvent necessary to dissolve the drug.

Radioimmunoassay. VP and OT concentrations in the perifusate fractions were determined by radioimmunoassay. The antisera utilized were generated in conjunction with Arnel Products (Brooklyn, NY). The antibodies (Ab) were used at final dilutions of 1:100,000-1:150,000 as provided appropriate assay sensitivity. The buffer for both OT and VP assays was 0.1 M PBS (pH 7.6) with 1 mg/ml bovine serum albumin and 1 mg/ml sodium azide. A 100- and a 50-µl aliquot of each fraction were assayed. The standards and samples were incubated for 72 h at 4°C in the presence of 5,000 counts/min of 125I-AVP or 96 h at 4°C with 3,500 counts/min 125I-oxytocin (New England Nuclear). Ab-bound VP and OT were separated from free hormone with dextran-coated charcoal. Radioactivity in the charcoal pellet was determined with a gamma-counter and converted to picograms per milliliter by comparison to a standard curve constructed from known concentrations of either OT or AVP. All samples from a given experiment were assayed at the same time. The minimum sensitivity was 1.0 pg/tube for VP and 0.5 pg/tube for OT.

RNA extraction and quantification. Total RNA was extracted by utilizing Tri-Reagent (Molecular Research Center, Cincinnati, OH) and assayed in a ribonuclease (RNase) protection assay utilizing a 32P-labeled full-length VP cRNA probe (680 bp) synthesized using the Promega Riboprotein System from the pGEM 4-AVP8C construct provided by T.G. Sherman, Georgetown University. A nucleic acid purification cartridge was used to isolate the radiolableled probe from nonspecific radioactivity. This full-length probe hybridized to both the VPmRNA and the OTmRNA because the full-length VPmRNA probe included the highly homologous region of neurophysin in VP and OT mRNA. After hybridization, the samples were treated with RNase to degrade any single-stranded RNA and the protected fragments were separated on a 1.3% agarose gel. Because the protected fragments from VP and OT mRNA were different sizes, it was possible to measure both OT and VP mRNA content with this assay. The details of the RNA protection assay have been described previously (41). After electrophoretic separation, the bands corresponding to VP and OT mRNA were cut from the gel and counted in liquid scintillation cocktail. Controls were included with standards of 0, 50, 100, 500, and 1,000 pg of sense VP mRNA generated from the cAVP-8C plasmid. RNA from a nonperifused explant and standard preparations of RNA from hydrated and dehydrated rats were included on each gel for comparison. Total extracted RNA from each explant was used in the hybridization reaction.

Data analysis. The results are expressed graphically as means of the percentage of basal release ± SE. Statistical significance was determined by ANOVA with repeated measures on log10 transformed data. When the ANOVA indicated statistical significance, simple mean effects analysis or Newman-Keuls individual mean comparisons were performed to determine if the difference between groups at specific time points was significant. The level of significance was set at P <=  0.05. On each figure, an arrow or a box depicts the time when the HNS explants were exposed to each agent, F values represent overall group differences, and symbols (such as *, +, etc.) indicate the probability that hormone release is different between means at individual time points.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Effect of KA and AMPA on VP and OT release. HNS explants were perifused with KA and AMPA in increasing concentrations at 2-h intervals (Figs. 1 and 2). As shown in Fig. 1, exposure to KA at 10 µM, 100 µM, and 1 mM increased VP release at all concentrations compared with the control group perifused with basal medium throughout (P < 0.05). However, in this sequential concentration-response paradigm, the VP response to 100 µM and 1 mM KA did not exceed that induced by 10 µM KA.


View larger version (28K):
[in this window]
[in a new window]
  		Fig. 1.   Effect of kainic acid (KA) on vasopressin (VP) release. VP release was significantly greater in KA ()-perifused explants compared with controls (). KA concentration was increased at 2-h intervals from 10 to 100 µM to 1mM as shown by boxes below graph. F value is between group comparison. Symbols (*, +) are level of significance between KA and controls at individual time points. Basal release: KA group, 27.4 ± 6.0 pg/ml; control group, 36.9 ± 7.1 pg/ml.



View larger version (30K):
[in this window]
[in a new window]
  		Fig. 2.   Effect of aminopropionic acid (AMPA) on VP and oxytocin (OT) release. A: AMPA significantly increased VP release at 10 and 100 µM. Peak release occurred at 10 µM. Subsequent exposure to a larger dose did not elicit a further increase in hormone release. Basal release: AMPA group, 17.8 ± 5.7 pg/ml; control group, 69.0 ± 18.0 pg/ml. B: AMPA significantly increased release of OT from hypothalamo-neurohypophysial (HNS) explants at all concentrations. Basal release: AMPA group, 87.9 ± 13.8 pg/ml; control group, 112.5 ± 18.9 pg/ml.

As shown in Fig. 2A, exposure to 10 µM AMPA resulted in a prominent increase in VP. The response decayed during the final hour of exposure to 10 µM AMPA, and a much smaller stimulation of VP release was observed during the subsequent exposure to 100 µM AMPA. However, when explants were exposed initially to 10 µM AMPA, the response was comparable to that elicited by 10 µM AMPA [for examples, see Fig. 9 and Ref. 38]. OT release was only measured in a subset of these explants. As seen in Fig. 2B, sustained stimulation of OT release was observed throughout exposure to all three concentrations of AMPA. Although initially the VP and OT responses to AMPA were similar, the VP response exhibited a greater tendency to decay. The sustained elevation of OT release required continued exposure to AMPA, as demonstrated in an experiment in which AMPA was withdrawn from the medium after a 2-h exposure. Both VP and OT release returned to basal within the time frame expected for washout of AMPA and the hormones from the perifusion chamber.

Role of AMPA receptor desensitization. CYC is a compound that specifically inhibits the desensitization of AMPA-type receptors. It was used to examine the contribution of AMPA-R desensitization to the decay in non-NMDA-R agonist-induced VP release. This compound was applied 30 min before application of the initial concentration of agonist and was present for the remainder of the experiment. The agonists were applied in the same manner as in the previous experiments. CYC significantly increased VP release during the initial exposure to 10 µM KA and AMPA (Fig. 3). However, the response rapidly decayed and became comparable to agonist alone at higher concentrations of agonists. These data support a role for AMPA-Rs in VP release induced by both KA and AMPA and suggest that AMPA-R desensitization attenuates VP release in response to both agonists. CYC applied alone had no effect on VP hormone release (data not shown, F = 3.044, P = 0.11).


View larger version (23K):
[in this window]
[in a new window]
  		Fig. 3.   Effect of cyclothiazide on KA and AMPA stimulation of VP release. Cyclothiazide (CYC) was applied 30 min before administration of KA (A) or AMPA (B). CYC, a drug that inhibits desensitization of AMPA-type receptors, significantly augmented VP release during exposure to lowest concentrations of KA and AMPA. Basal release: KA group, 27.4 ± 6.0 pg/ml; KA + CYC group, 18.91 ± 4.3 pg/ml; AMPA group, 17.8 ± 5.7 pg/ml; AMPA + CYC group, 17.0 ± 5.4 pg/ml.

Response to KA-R specific agonist SYM-2081. To determine if the stimulation of VP release by KA reflected activation of KA-Rs, explants were exposed to KA-R specific agonist SYM-2081. As shown in Fig. 4, SYM-2081 did not increase VP or OT release compared with the controls maintained on basal medium; however, subsequent exposure of the same explants to AMPA (100 µM) caused a significant increase in release of both VP and OT. For analysis of the response to AMPA exposure, basal release was recalculated as the release rate for each explant immediately before AMPA exposure (i.e., at 6 h).


View larger version (32K):
[in this window]
[in a new window]
  		Fig. 4.   Effect on VP and OT release of a KA-R specific agonist, SYM-2081 (0.5 µM), followed by AMPA (100 µM). Explants were exposed to SYM-2081 for 2 h. At the end of this period, perifusate was switched to one containing AMPA without SYM-2801. A: SYM-2081 () did not significantly alter VP release. Basal release was control group, 63.3 ± 26.1 pg/ml; SYM-2081 group, 74.3 ± 24.7 pg/ml. B: VP release significantly increased when SYM-2801 was replaced with AMPA () during last 2 h of experiment. Basal release: control group, 61.0 ± 27.1 pg/ml; AMPA group, 61.2 ± 24.9 pg/ml. (Basal release was adjusted at 6 h to analyze AMPA response.) C: SYM-2081 () did not significantly alter OT release. Basal release was: control group, 176.8 ± 80.5 pg/ml: SYM-2081, 189.9 ± 40.4 pg/ml. D: OT release was significantly increased during subsequent 2-h period when SYM-2081 was replaced by AMPA (). Basal release: control group, 184.8 ± 89.3 pg/ml; AMPA group, 110.2 ± 29.7 pg/ml. In this and in subsequent figures, arrows indicate time when explants were first exposed to agonists. Open symbols, controls.

Role of AMPA-Rs in osmotic stimulation of VP and OT release. To evaluate the role of AMPA-Rs in the VP and OT response to an osmotic stimulus, a specific AMPA-R antagonist, SYM-2206, was utilized. SYM-2206 is a substituted 1,2-dihydrophthalazine and acts as a noncompetitive inhibitor. An osmotic stimulus was created by gradually increasing the NaCl in the perifusion medium. The explants were exposed to a 6-h ramp increase in osmolality (30 mosmol/kgH2O over 6 h) in the presence or absence of SYM-2206 (6 µM, Fig. 5). In the presence of SYM-2206, there was a significant attenuation of both VP and OT release compared with explants exposed to the increase in osmolality alone. The antagonist alone did not affect VP or OT release compared with control explants exposed only to perifusion medium (VP: F = 0.087, P = 0.77; OT: F = 0.902, P = 0.53; data not shown). Some of the explants evaluated for hormone release were also examined for mRNA changes. In previous studies, exposure to the 6-h increase in osmolality resulted in an increase in VP and OT mRNA content of HNS explants (41, 47, 48). This osmotically induced increase in VP and OT mRNA was not observed in the presence of the AMPA-R antagonist, SYM-2206 (Fig. 6; VP: F = 27.7, P = 0.0062; OT: F = 11.24, P = 0.028).


View larger version (28K):
[in this window]
[in a new window]
  		Fig. 5.   Effect of SYM-2206, a specific AMPA receptor antagonist, on osmotically induced VP and OT release. Osmolality of the perifusion medium was gradually increased over a 6-h period by increasing NaCl concentration (Osm). A: VP release was significantly attenuated in the presence of SYM-2206. Basal release: Osm group (), 18.6 ± 3.6 pg/ml; SYM-2206 + Osm group (), 46.1 ± 14.1 p/ml-1. B: antagonist significantly attenuated osmotically induced OT. Basal release: Osm group (), 167.5 ± 30.6 pg/ml; Osm + SYM-2206 group (), 322.3 ± 122.8 pg/ml. Arrow indicates both the start of ramp increase in osmolality and the presence of SYM-2206.



View larger version (38K):
[in this window]
[in a new window]
  		Fig. 6.   Effect of SYM-2206 on osmotically stimulated VP and OT mRNA content. There was a significant inhibition of the osmotically induced increase in VP and OT mRNA with SYM-2206 (6 µM). *P < 0.03. Osm, stippled bars; Osm + SYM-2206, solid bars.

Effect of blocking non-NMDA-R on the response to NMDA. The observation that osmotic stimulation of hormone release can be blocked by either an AMPA-R antagonist (Fig. 5) or by NMDA-R antagonists (40) led to the hypothesis that glutamatergic stimulation of hormone release was dependent on coactivation of NMDA-R and non-NMDA-Rs. Because glutamate release by neurons endogenous to the explant might allow for coactivation of both receptor subtypes even during exposure to specific receptor agonists, the response to NMDA was evaluated in the presence of a non-NMDA-R antagonist. As shown in Fig. 7, NMDA (50 µM) significantly increased both VP and OT release from HNS explants compared with control groups. The VP response occurred earlier than the OT response. This may indicate different mechanisms of NMDA action on VP and OT release. NMDA-induced VP release was significantly inhibited in explants exposed to CNQX (10 µM) and NMDA simultaneously (Fig. 8A), but a significant increase in OT release remained in explants exposed to NMDA and CNQX compared with the control group receiving basal medium (Fig. 8B). OT release was not significantly different between the group receiving NMDA compared with the group exposed to NMDA and CNQX (Fig. 8C). These results indicate that NMDA-induced VP release requires activation of non-NMDA-Rs, because CNQX inhibited NMDA-induced VP release. In contrast, NMDA-induced OT release did not require activation of non-NMDA-Rs, but the OT release in both the NMDA group and the NMDA plus CNQX group was delayed relative to the VP response to NMDA alone.


View larger version (27K):
[in this window]
[in a new window]
  		Fig. 7.   Effect of NMDA on VP and OT release. A: NMDA significantly increased VP release. (F value indicates a difference between groups during 1st h of exposure to NMDA. ANOVA included only data from 4.75 to 5.75 h.) Basal release: control group, 123.2 ± 68.6 pg/ml; NMDA group, 127.2 ± 44.2 pg/ml. B: NMDA significantly increased OT release at this concentration. Basal release: control group, 254.3 ± 89.6 pg/ml; NMDA group, 194.5 ± 68.6 pg/ml.



View larger version (34K):
[in this window]
[in a new window]
  		Fig. 8.   Effect of CNQX on NMDA-induced VP and OT release. A: CNQX blocked the NMDA induced release of VP. (ANOVA was performed on data from 4.5 to 5.5 h. F value is difference between groups during this time.) Basal release: NMDA group, 127.2 ± 44.2 pg/ml; NMDA + CNQX group, 133.5 ± 69.7 pg/ml. B: NMDA significantly increased OT release in the presence of CNQX. Thus in contrast to VP release, NMDA-induced OT release is not blocked by CNQX. (F value is interaction between treatment and time.) Basal release: NMDA group, 193.5 ± 68.6 pg/ml; NMDA + CNQX group, 184.7 ± 85.8 pg/ml. C: there was no significant difference in NMDA-induced OT release in the presence or absence of CNQX. Basal release: NMDA + CNQX group, 184.7 ± 85.8 pg/ml; control group, 254.3 ± 89.6 pg/ml.

Effect of blocking NMDA-Rs on the response to KA and AMPA. To determine if activation of NMDA-Rs is required for the non-NMDA-R agonists to stimulate VP and OT release, the response to KA and AMPA was evaluated in the presence or absence of AP5, a competitive NMDA-R antagonist. The response of both VP and OT to KA (100 uM) and AMPA (100 uM) was not significantly attenuated by AP5 (100 uM). This concentration of AP5 was previously shown to attenuate osmotically stimulated VP release without altering basal VP release from HNS explants (40). AP5 did not alter the response to KA. There was a significant increase in VP (F = 8.789, P < 0.0001) and OT release (F = 5.408, P < 0.001) in response to KA, but the response was not significantly different in the presence and absence of AP-5. The VP response to KA was comparable to that shown in Fig. 1. Similarly for the AMPA experiments, VP release changed significantly over time in both the AMPA and AMPA plus AP5 groups (Fig. 9; F=5.96; P < 0.0001), but the response was not different in the presence or absence of AP5 (VP: F = 0.00; P = 1.0; OT: F = 0.25, P = 0.62). This indicates that AMPA-induced release is independent of NMDA-R activation.


View larger version (18K):
[in this window]
[in a new window]
  		Fig. 9.   Effect of AP5 on AMPA-induced VP release. VP response to AMPA was not significantly altered by AP5 (group F value shown in figure), but there was a significant change in release over time for both groups (F = 5.96, P < 0.0001) indicative of AMPA-induced VP release. Basal release was: AMPA group () 42.3 ± 12.3 pg/ml; AMPA + AP5 group () 114.3 ± 58.7 pg/ml.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

VP and OT release from the hypothalamic supraoptic-neurohypophysial pathway is controlled by a large number of synaptic inputs involving a wide variety of neurotransmitters (33). Glutamate is one of the primary excitatory agents, with 20-22% of the nerve terminals in the main body of SON and 38% of the terminals in the ventral dendritic neuropil being glutamatergic (18). A variety of techniques has been used to study the influence of glutamate on VP and OT neurons, but direct evaluation of the respective roles of the non-NMDA-Rs, the KA-Rs and AMPA-Rs, on the final output of these neurons, VP and OT release, had not been assessed. In addition, their roles in transducing osmotic information had not been assessed. These issues were addressed in the current studies.

Both non-NMDA-R agonists, KA and AMPA, stimulated the release of VP and OT. Because KA and AMPA are relatively nonspecific agonists with each being able to activate the other group of receptors at certain concentrations, attention was paid to discerning the relative contribution of the two-receptor groups to the observed responses. AMPA induced hormone release at concentrations consistent with specific activation of AMPA-Rs. The EC50 at GluR1-3 for AMPA ranges from 1.3 µM for GluR1 to 36 µM for GluR3 (13). In contrast the KA-R, GluR5, has an EC50 value of 3 mM for AMPA. The discriminating range for KA at KA and AMPA-Rs is not as great, with the KA EC50 values ranging from 39 to 130 µM for the AMPA-Rs, GluR1-4, and 1.5 to 33.6 µM for the KA-Rs (13). Therefore, it is likely that the activation of hormone release by both KA and AMPA can be attributed to activation of AMPA-Rs that are expressed in SON.

Additional evidence that supports this conclusion includes: 1) the potentiation of both KA and AMPA effects by CYC, and 2) the ineffectiveness of a KA-R specific agonist. Because CYC specifically blocks the desensitization of AMPA-Rs (25), its potentiation of the KA response suggests that KA is activating (and desensitizing) AMPA-Rs. The lack of response to the KA-R specific agonist also suggests that the response to KA reflects activation of AMPA-Rs. In vitro, this compound demonstrated a 500- to 2,000-fold selectivity for the KA-Rs, GluR5 and 6, vs. the AMPA-Rs, GluR1-3 (9), and it had an EC50 value of 0.12 µM for GluR5 and 0.23 µM for GluR6. Because the current experiments were performed at five times the EC50 for SYM-2081, this concentration should have elicited a response if KA-Rs are important contributors to VP and OT release. Furthermore, although SYM-2081 did not elicit hormone release, AMPA subsequently elicited a significant increase in VP and OT, indicating that the explants were capable of being stimulated. These observations support the interpretation that KA stimulation of VP and OT release reflects activation of AMPA-Rs and corresponds with morphological reports of only weak expression in SON and hypothalamus of only two of the KA-R subunits, GluR5 and GluR6 (27, 43). However, under other physiological conditions, expression of these receptors may be upregulated and they may become more important in the regulation of hormone release.

Hormone release was not sustained in response to extended exposure to either KA or AMPA. The ability of CYC to potentiate responses to KA and AMPA indicated that receptor desensitization was involved, and previous studies had demonstrated that receptor desensitization completely eliminated detectable effects of glutamate on VP release from HNS explants (35). However, even in the presence of CYC, the responses to AMPA and KA were not sustained. Because the process of desensitization has been postulated to contribute to the termination of glutamatergic activation at these receptors (42), desensitization may be important in preventing excitotoxic damage of the neurons during exposure to glutamate. Thus in the presence of CYC, excitotoxicity or other processes, such as depolarization blockade or fatique of stimulus-secretion coupling, may limit more extended responses to AMPA or KA. Another possibility is that activation of GluRs on local inhibitory neurons limits the response to AMPA or KA (6). In previous studies, bicuculline, an inhibitor of GABA receptors, increased VP release from HNS explants (34). Therefore, local GABAergic circuits are tonically active in these explants. This may also be the reason that basal release was not decreased by CNQX.

Another goal of this study was to evaluate the interactions between NMDA-Rs and non-NMDA-Rs in eliciting VP and OT release. These studies led to the interesting observation that VP and OT release were differentially affected by NMDA. The VP response was rapid in onset and not sustained, whereas the OT response developed slowly and was sustained. This may reflect differential expression of NMDA-R (NR) subunits in VP and OT neurons or differences in activation of local circuitry regulating these two neuronal populations. Relative to the first suggestion, NR1 subunits are expressed equally in VP and OT neurons, but NR2 subunit expression differs. NR2C expression is fivefold greater in VP neurons than in OT neurons (1). This could confer different functional properties to the NMDA-Rs. In expression systems, the combination of NR1 with NR2C vs. NR2B subunits results in altered sensitivity to magnesium blockade and affinity for glycine. Receptors consisting of NR1 in combination with NR2A or 2B demonstrate a stronger blockade by magnesium ions compared with combinations with NR2C or D (20). As for glycine affinity, NR1 subunits in combination with NR2C possess a higher affinity for glycine (46). The differential subunit expression could result in VP neurons being more easily activated by NMDA and could account for the earlier VP response to NMDA compared with the OT release pattern. Reports on the action of NMDA on OT neurons are mixed. In one report, NMDA did not elicit activation of OT magnocellular neurons, and in another, non-NMDA-Rs were reported to be more important than NMDA-Rs on OT neurons (22, 30). However, these reports contrast with that of Stern et al. (39) that the NMDA-mediated miniature excitatory postsynaptic currents displayed by VP and OT neurons had similar amplitude and kinetic properties. In an additional report, NMDA-R activation induced rhythmic bursting activity in all SON neurons (14), and both VP and OT neurons responded with increased activation from NMDA application (15). The suggestion that the diverse OT and VP responses to NMDA reflect activation of local circuits specific for VP and OT neurons is based on the existence of substantial evidence for glutamate activation of both excitatory and inhibitory afferents to magnocellular neurons in SON and paraventricular nucleus (5, 6). However, to date, the possibility that these circuits differentiate between OT and VP neurons has not been evaluated.

The inhibition of NMDA-induced VP release by CNQX is consistent with the hypothesis that AMPA-mediated membrane depolarization is required to remove the magnesium blockade of NMDA receptors. This corresponds with the in vivo finding that intracerebroventricular injection of CNQX blocked an antidiuretic response to intracerebroventricular administration of NMDA (4). Two doses of 40 nmol of CNQX were injected over a 15-min period with the application of the agonist 5 min after the second injection. The amount of NMDA (1-4 nmol) injected elicited an effect when applied alone (4). In another study, CNQX (1 mM) caused a significant decrease in the amplitude of NMDA-evoked bursts recorded in SON neurons in vivo (22).

The unexpected finding was that CNQX did not also block NMDA-induced OT release. However, as discussed above, NMDA elicited a slow increase in OT release suggestive of an indirect effect. Another possibility is that the role of AMPA-Rs is different in VP and OT neurons. According to Stern et. al. (39), there is a differential response to activation of AMPA-Rs between OT and VP neurons with the high-frequency summation of AMPA-R-mediated excitatory potentials being smaller in OT neurons. This may make OT neurons less dependent on AMPA-Rs for eliciting hormone release. It also may explain the somewhat smaller effect of AMPA on OT release compared with VP release (e.g., Fig. 2).

The failure of NMDA-R blockade to prevent responses to AMPA or KA was also unexpected. Because NMDA-Rs mediate rhythmic bursting activity in VP neurons and because an intermittent firing pattern is important for preventing fatigue of stimulus-secretion coupling and thus for maintaining sustained hormone release (3), it was predicted that AP5, the NMDA-R antagonist, would inhibit AMPA-induced VP but not OT release. However, the current studies indicate that AMPA-mediated stimulation of both VP and OT release can occur independently of NMDA-Rs. These results concur with the in vivo observation that AP5 did not block the antidiuretic response to the intracerebroventricular application of AMPA (4). Thus the present and previous results indicate that NMDA-R activation is not required for AMPA- or KA-induced VP release.

In conclusion, these results demonstrate that release of VP and OT is stimulated by AMPA-R activation and that both AMPA-Rs and NMDA-Rs are essential for osmotic stimulation of hormone release. However, this dependence of osmotic stimulation on activation of both receptor types does not reflect a requirement for coactivation of these receptors to generate firing patterns consistent with sustained hormone release.

Perspectives

Studies designed to elucidate the mechanisms involved in the osmotic regulation of VP release have spanned the decades since Verney originally postulated the existence of osmoreceptors in 1947 (45). The cumulative evidence since that time supports a multicomponent sensing system in which both the magnocellular VP and OT neurons and neurons in the OVLT express stretch-inactivated cation channels that result in neuronal depolarization in response to increases in osmolality of the extracellular fluid. Communication between the osmosensitive neurons in OVLT and the magnocellular neurons is required to achieve the sensitive osmotic regulation of hormone release observed physiologically (see Refs. 7 and 33 for review). Thus characterization of the chemical transmission between these neurons has received considerable attention. Glutamate has been identified as a transmitter in this pathway (29), but prior studies indicated that other transmitters/receptors (e.g., angiotensin II and acetylcholine; Refs. 36 and 37) are also critical for osmotic regulation of VP release. It is unlikely that angiotensin II and acetylcholine serve as cotransmitters with glutamate from the OVLT. Angiotensin is produced in neurons that innervate SON, including the osmoregulatory pathway from the subfornical organ, but it is not present in OVLT neurons (16). However, it is produced in the magnocellular neurons (16), and therefore, it may serve an autocrine role in the regulation of VP and OT release from HNS explants. Recently, angiotensin II and two other excitatory peptides have been shown to activate the stretch-inactivated ion channel responsible for osmosensitivity in the magnocellular neurons and osmoreceptive elements in OVLT and to potentiate the response of these channels to hypertonicity (8). Thus autocrine release of angiotensin II by the SON neurons may be critical in eliciting responses to osmotic stimulation. A role for acetylcholine is less clear. Although the SON richly expresses alpha 7-nicotinic (bungarotoxin) receptors, only minimal innervation of SON by cholinergic fibers has been observed (see Ref. 33). Thus acetylcholine may not be the endogenous ligand for these receptors, but activation of these ionotropic receptors may increase excitability of the SON neurons, rendering them capable of responding to osmotic stimulation. Additional work is required to understand how these mechanisms interact with glutamatergic membrane depolarization to induce osmotic regulation of VP and OT release.


    ACKNOWLEDGEMENTS

This work was supported by National Institutes of Health Grant RO1-NS-27975.


    FOOTNOTES

Address for reprint requests and other correspondence: C. D. Sladek, Dept. of Physiology, Chicago Medical School, North Chicago, IL 60064 (E-mail: sladekc{at}finchcms.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 30 May 2000; accepted in final form 8 September 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Al-Ghoul, WM, Meeker RB, and Greenwood RS. Differential expression of five N-methyl-D-aspartate receptor subunit mRNAs in vasopressin and oxytocin neuroendocrine cells. Mol Brain Res 44: 262-272, 1997[Medline].

2.   Armstrong, WE, and Stern JE. Electrophysiological and morphological characteristics of neurons in perinuclear zone of supraoptic nucleus. J Neurophysiol 78: 2427-2437, 1997[Abstract/Free Full Text].

3.   Bicknell, RJ, Brown D, Chapman C, Hancock PD, and Leng G. Reversible fatigue of stimulus-secretion coupling in the rat neurohypophysis. J Physiol (Lond) 348: 601-613, 1984[Abstract/Free Full Text].

4.   Bisset, GW, and Fairhall KM. Release of vasopressin and oxytocin by excitatory amino acid agonists and the effect of antagonists on release by muscarine and hypertonic saline, in the rat in vivo. Br J Pharmacol 117: 309-314, 1996[ISI][Medline].

5.   Boudaba, C, Schrader LA, and Tasker JG. Physiological evidence for local excitatory synaptic circuits in the rat hypothalamus. J Neurol 77: 3396-3400, 1997.

6.   Boudaba, C, Szabo K, and Tasker JG. Physiological mapping of local inhibitory inputs to the hypothalamic paraventricular nucleus. J Neurosci 16: 7151-7160, 1996[Abstract/Free Full Text].

7.   Bourque, CW, Oliet SHR, and Richard D. Osmoreceptors, osmoreception, and osmoregulation. Front Neuroendocrinol 15: 231-274, 1994[ISI][Medline].

8.   Chakfe, Y, and Bourque CW. Excitatory peptides and osmotic pressure modulate mechanosensitive cation channels in concert. Nat Neurosci 3: 572-579, 2000[ISI][Medline].

9.   Donovan, SD, Beg A, Gunther JM, and Twyman RE. The methylglutamate, SYM 2081, is a potent and highly selective agonist at kainate receptors. J Pharmacol Exp Ther 285: 539-545, 1998[Abstract/Free Full Text].

10.   Ginsberg, SD, Price DL, Blackstone CD, Huganir RL, and Martin LJ. The AMPA glutamate receptor GluR3 is enriched in oxytocinergic magnocellular neurons and is localized at synapses. Neuroscience 65: 563-575, 1995[ISI][Medline].

11.   Gribkoff, VK, and Dudek FE. The effects of the excitatory amino acid antagonist kynurenic acid on synaptic transmission to supraoptic neuroendocrine cells. Brain Res 442: 152-156, 1988[ISI][Medline].

12.   Hattori, Y, Shibuya I, Tanaka K, Kabashima N, Ueta Y, and Yamashita H. Ionotropic and metabotropic glutamate receptor agonist-induced [Ca2+]i increase in isolated rat supraoptic neurons. J Neuroendocrinol 10: 383-389, 1998[ISI][Medline].

13.   Hollman, M, and Heinemann S. Cloned glutamate receptors. Annu Rev Neurosci 17: 31-108, 1994[ISI][Medline].

14.   Hu, B, and Bourque CW. NMDA receptor-mediated rhythmic bursting activity in rat supraoptic nucleus neurones in vitro. J Physiol (Lond) 458: 667-687, 1992[Abstract/Free Full Text].

15.   Hussy, N, Boissin-Agasse L, Richard P, and Desarmenien MG. NMDA receptor properties in rat supraoptic magnocellular neurons: characterization and postnatal development. Eur J Neurosci 9: 1439-1449, 1997[ISI][Medline].

16.   Lind, RW, Swanson LW, and Ganten D. Organization of angiotensin II-immunoreactive cells and fibers in the rat central nervous system. An immunohistochemical study. Neuroendocrinology 40: 2-24, 1985[ISI][Medline].

17.   Martin, D, Blackstone CD, Levey AI, Huganir RL, and Price DL. AMPA glutamate receptor subunits are differentially distributed in rat brain. Neuroscience 53: 327-358, 1993[ISI][Medline].

18.   Meeker, RB, Swanson DJ, Greenwood RS, and Hayward JN. Quantitative mapping of glutamate presynaptic terminals in the supraoptic nucleus and surrounding hypothalmus. Brain Res 600: 112-122, 1992.

19.   Meeker, RB, Swanson DJ, and Hayward JN. Light and electron microscopic localization of glutamate immunoreactivity in the supraoptic nucleus of the rat hypothalamus. Neuroscience 33: 157-167, 1989[ISI][Medline].

20.   Monyer, H, Burnasherv N, Laurie DJ, Sakmann B, and Seeburg PH. Developmental and regional expression in the rat brain and functional properties of four NMDA receptors. Neuron 12: 529-540, 1994[ISI][Medline].

21.   Nissen, R, Hu B, and Renaud LP. N-methyl-D-aspartate receptor antagonist ketamine selectively attenuates spontaneous phasic activity of supraoptic vasopressin neurons in vivo. Neuroscience 59: 115-120, 1994[ISI][Medline].

22.   Nissen, R, Hu B, and Renaud LP. Regulation of spontaneous phasic firing of rat supraoptic vasopressin neurones in vivo by glutamate receptors. J Physiol (Lond) 484: 415-424, 1995[ISI][Medline].

23.   Nowak, L, Bregostovski P, Ascher P, Herbert A, and Prochiantz A. Magnesium gates glutamate-active channels in mouse central neurones. Nature 307: 462-465, 1984[Medline].

24.   Partin, KM, Bowie D, and Mayer ML. Structural determinants of allosteric regulation in alternatively spliced AMPA receptors. Neuron 14: 833-843, 1995[ISI][Medline].

25.   Partin, KM, Patneau DK, Winters CA, Mayer ML, and Buonanno A. Selective modulation of desensitization at AMPA versus kainate receptors by cyclothiazide and concanavalin A. Neuron 11: 1069-1082, 1993[ISI][Medline].

26.   Pelletier, JC, Hesson DP, Jones KA, and Costa AM. Substituted 1,2-dihydrophthalazines: potent, selective, and noncompetitive inhibitors of the AMPA receptor. J Med Chem 39: 343-346, 1996[ISI][Medline].

27.   Petralia, RS, Wang YX, and Wenthold RJ. Histological and ultrastructural localization of the kainate receptor subunits, KA2 and GluR6/7, in the rat nervous system using selective antipeptide antibodies. J Comp Neurol 349: 85-110, 1994[ISI][Medline].

28.   Petralia, RS, and Wenthold RJ. Light and electron immunocytochemical localization of AMPA-selective glutamate receptors in the rat brain. J Comp Neurol 318: 329-354, 1992[ISI][Medline].

29.   Richard, D, and Bourque CW. Synaptic activation of rat supraoptic neurons by osmotic stimulation of the organum vasculosum lamina terminalis. Neuroendocrinology 55: 609-611, 1992[ISI][Medline].

30.   Richardson, CM, and Wakerly JB. Glutamate excitation of oxytocin neurones in vitro involves predominantly non-NMDA receptors. Brain Res 767: 158-161, 1997[ISI][Medline].

31.   Sato, K, Kiyama H, and Tohyama M. The differential expression patterns of messenger RNAs encoding non-N-methyl-D-aspartate glutamate receptor subunits (GluR1-4) in the rat brain. Neuroscience 52: 515-539, 1993[ISI][Medline].

32.   Sato, K, Mick G, Kiyama H, and Tohyama M. Expression patterns of a glutamate-binding protein in the rat central nervous system: comparison with N-methyl-D-aspartate receptor subunit 1 in rat. Neuroscience 64: 459-475, 1995[ISI][Medline].

33.   Sladek, CD. Antidiuretic hormone:synthesis and release. In: Handbook of Physiology: The Endocrine System, edited by Fray JCS. Bethesda, MD: Am Physiol Soc, 2000, sec 7, vol III, p. 436-495.

34.   Sladek, CD, and Armstrong WE. GABA antagonists stimulate vasopressin release from organ-cultured hypothalamo-neurohypophyseal explants. Endocrinology 120: 1576-1580, 1987[Abstract].

35.   Sladek, CD, Badre SE, Morsette DJ, and Sidorowicz HE. Role of non-NMDA receptors in osmotic and glutamate stimulation of vasopressin release: effect of rapid receptor desensitization. J Neuroendocrinol 10: 897-903, 1998[ISI][Medline].

36.   Sladek, CD, and Joynt RJ. Cholinergic involvement in osmotic control of vasopressin release by the organ cultured rat hypothalamo-neurohypophyseal system. Endocrinology 105: 367-371, 1979[Abstract].

37.   Sladek, CD, and Joynt RJ. Role of angiotensin in the osmotic control of vasopressin release by the organ-cultured rat hypothalamo-neurohypophyseal system. Endocrinology 106: 173-178, 1980[Abstract].

38.   Sladek, CD, Swenson KL, Kapoor R, and Sidorowicz HE. The role of steroid hormones in the regulation of vasopressin and oxytocin release and mRNA expression in hypothalamo-neurohypophysial explants from the rat. Exp Physiol 85S: 171S-177S, 2000[Abstract].

39.   Stern, JE, Galarreta M, Foehring RC, Hestrin S, and Armstrong WE. Differences in the properties of ionotropic glutamate synaptic currents in oxytocin and vasopressin neuroendocrine neurons. J Neurosci 19: 3367-3375, 1999[Abstract/Free Full Text].

40.   Swenson, KL, Badre SE, Morsette DJ, and Sladek CD. N-methyl-D-aspartic (NMDA) stimulation of vasopressin release: role in osmotic regulation and modulation by gonadal steroids. J Neuroendocrinol 10: 679-685, 1998[ISI][Medline].

41.   Swenson, KL, and Sladek CD. Gonadal steroid modulation of vasopressin secretion in response to osmotic stimulation. Endocrinology 138: 2089-2097, 1997[Abstract/Free Full Text].

42.   Trussell, LO, and Fischbach GD. Glutamate receptor desensitization and its role in synaptic transmission. Neuron 3: 209-218, 1998.

43.   Van den Pol, AN, Herman-Borgmeyer I, Hofer M, Ghosh P, and Heinemann S. Ionotropic glutamate-receptor gene expression in hypothalamus: localization of AMPA, kainate, and NMDA receptor RNA with in situ hybridization. J Comp Neurol 343: 428-444, 1994[ISI][Medline].

44.   Van den Pol, AN, Waurin JP, and Dudek FE. Glutamate, the dominant excitatory transmitter in neuroendocrine regulation. Science 250: 1276-1278, 1990[Abstract/Free Full Text].

45.   Verney, EB. The antidiuretic hormone and the factors which determine its release. Proc R Soc Lond B Biol Sci 135: 25-106, 1947.

46.   Wafford, KA, Bain CJ, Le BB, Whiting PJ, and Kemp JA. Preferential co-assembly of recombinant NMDA receptors composed of three different subunits. Neuroreport 4: 1347-1349, 1993[ISI][Medline].

47.   Yagil, C, and Sladek CD. Effect of extended exposure to hypertonicity on vasopressin mRNA content in hypothalamo-neurohypophyseal explants. Endocrinology 127: 1428-1435, 1990[Abstract].

48.   Yang, CR, Senatorov VV, and Renaud LP. Organum vasculosum lamina terminalis-evoked post-synaptic responses in rat supraoptic neurones in vitro. J Physiol (Lond) 477: 59-74, 1994[ISI][Medline].

Am J Physiol Regul Integr Comp Physiol 280(2):R313-R322
0363-6119/01 $5.00 Copyright © 2001 the American Physiological Society


This article has been cited by other articles:


Home page
 Exp. Biol. Med.Home page
N. F. Rossi and H. Chen
Aminopropionic Acid receptors in paraventricular nucleus mediate pressor and vasopressin responses to endothelin-1 in subfornical organ.
Experimental Biology and Medicine, June 1, 2006; 231(6): 1075 - 1080.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Regul. Integr. Comp. Physiol.Home page
T. E. Lohmeier
Neurohypophysial hormones
Am J Physiol Regulatory Integrative Comp Physiol, October 1, 2003; 285(4): R715 - R717.
[Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (5)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Morsette, D. J.
Right arrow Articles by Sladek, C. D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Morsette, D. J.
Right arrow Articles by Sladek, C. D.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online