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Department of Cell Biology and Neuroscience, University of California, Riverside, California
Submitted 9 December 2003; accepted in final form 6 September 2004
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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-S infusion into recorded neurons. Under these conditions, the adenosinergic inhibition of firing and reduction of spike duration were blocked, suggesting the effects were mediated by postsynaptic adenosine receptors. That the effects on excitability could be due to direct activation of adenosine A1 receptors on supraoptic neurons was further explored immunocytochemically via the co-labeling of magnocellular neurons with polyclonal antibodies raised against the A1 receptors. It is concluded that adenosine, acting at postsynaptic A1 receptors, exhibits a powerful inhibitory influence on supraoptic magnocellular activity and is an important endogenous regulator of magnocellular neuroendocrine function. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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). Neuroeffector molecules influencing neuronal firing activity thus have the potential for pronounced downstream effects on general animal physiology.
SON and PVN neurons can be spontaneously active both in vivo and in vitro, and several neurotransmitters not only modulate this activity (Armstrong 1995; Li and Hatton 1996; Shibuya et al. 2000), but also control OT and VP release from the neurohypophysis (Sladek and Kapoor 2001; Wang et al. 2002). Recordings from horizontal and coronal brain slices containing the SON show an abundance of postsynaptic potentials (PSPs), mediated largely via cation-fluxing glutamate receptors or GABAA receptors. Along with glutamate and GABA, many other neuromodulatory systems are known to project to the SON (Hatton 1990) and effect magnocellular physiology. One neuromodulator shown to affect fast neurotransmission in the SON is adenosine (Oliet and Poulain 1999).
Adenosine is found throughout the mammalian brain. It is currently thought to have four receptor subtypes (A1, A2A, A2B, and A3), all linked to G proteins (Fredholm et al. 2001). Basal extracellular adenosine concentrations range from 40 to 460 nM (Ballarin et al. 1991; Latini and Pedata 2001), with local synaptic concentrations being higher. Although specific adenosine projections to the SON have not been shown, tuberomammillary nucleus fibers, which project to the SON (Weiss et al. 1989), are enriched in adenosine deaminase (Senba et al. 1985; Staines et al. 1987), a marker for adenosinergic neurons. Additionally, adenosine is formed via extracellular catabolism of adenine nucleotides (e.g., ATP), found in several vesicle types and released along with the other contents of vesicle (Jo and Role 2002; Sperlagh et al. 1998). A transmitter in its own right in the SON and elsewhere (Hiruma and Bourque 1995; Poelchen et al. 2001; Shibuya et al. 1999), ATP is rapidly converted to adenosine in the synaptic cleft (Dunwiddie et al. 1997).
Specific ligands and antibodies have been employed to describe the distribution of the A1 and A2A receptor subtypes in the nervous system, including the SON. A recent study using RT-PCR techniques has provided evidence for transcripts of all four subtypes in the SON (Noguchi and Yamashita 2000) and electrophysiological evidence for an adenosine A1 receptor mediated reduction in Ca2+ currents in dissociated SON neurons. However, both an adenosinergic influence on excitability and direct anatomical evidence for postsynaptic adenosine receptors in the SON is still lacking. Here, we provide whole cell patch-clamp and confocal microscopic evidence for exogenous and endogenous effects of adenosine on SON neuronal excitability via postsynaptic A1 receptors.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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All salts used in the perfusion media and whole cell recording solution and were purchased from Fisher Scientific (Fair Lawn, NJ). Adenosine, the A1 agonists N6-cyclopentyladenosine (CPA) and cyclohexyladenosine (CHA), the A1 antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), and the A2 agonist 5'-(N-cyclopropyl) carboxamidoadenosine (CPCA) were purchased from RBI/Sigma (St. Louis, MO). EGTA, HEPES, GDP--S, and 3(N-morpholino) propanesulfonic acid (MOPS) were also purchased from RBI/Sigma. The adenosine uptake inhibitor Dilazep was purchased from Tocris Cookson (Ellisville, MO).
Slice preparation and procedures
Slices were prepared similarly to those previously described (Yang and Hatton 1997) and were in accordance with the University of California, Riverside animal handling and use guidelines. Briefly, adult male Sprague-Dawley rats (45–70 days old) were decapitated, and the brains were rapidly removed and placed in ice-cold oxygenated artificial cerebral spinal fluid (ACSF) consisting of (in mM) 126 NaCl, 5 KCl, 1.25 NaH2PO4, 1.3 MgSO4, 10 D-glucose, 26 NaHCO3, 2.4 CaCl2, and 5 MOPS, pH 7.4. The combination of this organic buffer and NaHCO3 has been found in earlier studies to better stabilize the pH over prolonged recording sessions than does the use of the bicarbonate buffer alone. Brains were placed ventral side up, blocked for slicing, and glued via the dorsal cerebrum to a specimen holder of a Vibratome. Gassed (95% O2-5% CO2), ice-cold ACSF bathed the brain block, and a single horizontal hypothalamic slice containing the supraoptic nucleus (400–500 µm thick) was cut and placed in a bath of gassed ACSF for slice bisection and cropping. One hemi-slice was placed in a recording chamber held at 35°C and the other in a holding chamber at room temperature.
Whole cell recording
Procedures were similar to those described previously (Li et al. 1995). Patch electrodes (borosilicate, 1–2 µm tip diam) were pulled using a multi-stage pipette puller (Sutter Instruments) and filled with a recording solution consisting of (in mM) 130 K+-gluconate, 10 KCl, 2 MgCl2, 10 HEPES, 0.4 EGTA, 2 K2-ATP, and 0.4 Na2-GTP, and 0.08% (wt/vol) Lucifer yellow (potassium salt). The final pH and osmolality of the recording solution was 7.3 (adjusted with KOH) and 294 ± 3 mOsm/kg (adjusted with H2O), respectively. The final DC resistance of the pipettes was 3–5 M
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Patch pipettes were guided to cells visualized under near infrared differential interference contrast video microscopy (Leica DMLFSA equipped with a Dage IR-1000 camera), and gigaohm seals were obtained. Brief suction resulted in the establishment of whole cell configuration, and the bridge circuitry of the amplifier (Axoclamp 2-B, Axon Instruments) was immediately engaged and optimized. Following this, a liquid junction potential correction of 
7 mV was subtracted on-line from the recorded resting potential. In voltage-clamp experiments, cells were clamped at either –70 or –35 mV to monitor excitatory and inhibitory postsynpatic currents (EPSCs and IPSCs), respectively. Several cells were tested for a sustained outward rectification current (SOR), an electrophysiological property useful in distinguishing cells that synthesize OT from those synthesizing VP (Armstrong and Stern 1998; Stern and Armstrong 1997). Cells were voltage-clamped at –70 mV and treated with serial voltage steps from –40 to –110 mV. A clear example of the presence (Fig. 2E, inset) or absence (Fig. 1C, inset) of a SOR was rare, which may be due to our use of male rats and moderately elevated extracellular K+ ([K+]o).
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Immunocytochemistry
Adult male rats were deeply anesthetized and transcardially perfused with 4% paraformaldehyde. Coronal sections (thickness = 50 µm) were cut using a Vibratome and stored in 24-well culture plates. All immunocytochemical reactions were done in the wells. Sections were washed in PBS and treated with 0.3% Triton X-100 for 30 min. Following a PBS wash, sections were incubated in a 10% normal goat serum blocking buffer for 30 min. All antibodies were diluted in blocking buffer. Monoclonal antibodies to oxytocin (PS-38) and vasopressin (PS-41), a gift from Dr. H. Gainer (NINDS), were combined with rabbit polyclonal antibodies against the adenosine A1 receptor (Sigma or Alpha Diagnostics) The dilutions used were 1:400 for both PS-38 and -41 and 1:50 (Sigma) or 1:100 (ADI) for the A1 receptor antibody. Incubation in primary antibody solutions were performed at room temperature for 4 h. Following several PBS washes, the sections were incubated in blocking buffer for 30 min, followed by secondary antibody solutions for 2 h at room temperature. Secondary antibodies from Molecular Probes (Eugene, OR) were goat anti-rabbit Alexa Flour 546 and goat anti-mouse Alexa Flour 488. Both were used in dilutions of 1:1,000. Control experiments with the absence of primary antibody were done in parallel. Images of the sections were collected on a confocal microscope (Leica SP2) under sequential scan mode. Data were collected with a pinhole set to 1 airy unit corresponding to optical sections of <300 nm. Images are z-series projections with a depth difference of 
0.5 µm between successive sections.
Data analysis
All electrophysiological data were digitized at 10 kHz, filtered at 5 kHz, and collected for off-line analysis using pClamp software (Axon Instruments). Statistical analysis using SigmaStat software consisted of the paired or unpaired Student's t-test or their nonparametric equivalents. Minimal statistical significance was taken as P < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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Data from 81 recorded cells were used in this study. As measured 5 min following whole cell establishment, cells included in our analyses had input resistances ranging from 0.3 to 0.9 G, action potential amplitudes > 65 mV, displayed spontaneous PSPs, and resting membrane potentials between –52 and –62 mV. These membrane features are comparable with numerous published studies using this system. All cells either fired action potentials spontaneously or in response to current injection of <10 pA. The firing rate across all cells averaged 3.09 ± 0.27 Hz during the second minute of recording at resting potential.
To investigate the effects of brief ligand exposure, initial experiments used bolus application of adenosine. A high concentration of the ligand (prepared in ACSF) was added directly to the drip-bag, resulting in an estimated bath concentration of 10–4 M. This induced a hyperpolarization in all tested cells (–4.50 ± 1.26 mV), resulting in suppressed firing activity persisting for 3–10 min. Perfusion of increasing adenosine concentrations was performed on six cells to determine the effective concentrations for the inhibition of firing activity. Adenosine (100 nM to 100 µM) resulted in an EC50 of 10 µM for the adenosine-induced cessation of firing. Whereas 100 nM was not effective in abolishing firing, one cell responded to 1 µM, three stopped firing in 10 µM (Fig. 1, B and C), and all six cells tested were silenced in the presence of 100 µM adenosine. Bath application of 100 µM adenosine induced a response similar to, although stronger than, that seen with bolus application, a hyperpolarization (–6.08 ± 0.83 mV) and termination of action potential firing.
Adenosine affects action potential duration
To investigate the effect of adenosine on action potential frequency and duration, measurements were taken from an average of 10 action potentials just prior to the adenosine-induced hyperpolarization and cessation and compared with control values from the same firing episode measured 1 min before adenosine application. Measurements regarding duration were made at half-maximum amplitude and represent an average of 10 spikes. In all cases, action potentials measured in the presence of adenosine were shorter in duration than those recorded in the control period (Fig. 2, A and C; n = 11 cells). A closer examination of the action potential in nine cells revealed that the K+-mediated falling slope was increased in eight of nine cases (Fig. 2A). The adenosinergic reduction of action potential duration averaged –134 ± 41 µs, a 7.6 ± 2.2% reduction (P < 0.01) from control values (Fig. 2C). Prior to the termination of firing, spike frequency was also reduced causing mean firing levels in these cells to fall from 2.84 ± 0.30 to 1.25 ± 0.20 Hz (n = 7; Fig. 2D).
Involvement of the A1 receptor
The next important task was to determine which adenosine receptor subtypes were mainly responsible for the observed effects. Because adenosine A1 receptor activation is known to hyperpolarize cells by opening a K+ channel, which is consistent with our observed effects, we first determined the effects of A1 receptor-related compounds. DPCPX is a well-tested, selective antagonist of adenosine A1 receptors (Lohse et al. 1987). Incubation of the slice with DPCPX (1 µM) prevented the effects of adenosine (Fig. 3, Aa and Ab). The effects induced by both CPA and CHA (0.1–1 µM), selective A1 receptor agonists, mimicked those of adenosine. Both CPA and CHA hyperpolarized the resting membrane potential by –6.55 ± 1.27 mV (P < 0.001) and reduced action potential duration (–240 ± 111 µs, P < 0.01; Figs. 3, D and E, and 4B). In most cases (7 of 9), the A1 receptor agonists increased the slope of the K+-mediated falling phase of the action potential (Fig. 3F) by an average of –10.68 ± 3.25 mV/ms. Like adenosine, the A1 agonists also reduced firing frequencies from 2.84 ± 0.30 to 1.25 ± 0.40 Hz (P < 0.02, Fig. 3C).
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). Several studies have investigated neuromodulatory actions of adenosine A2 receptors, especially the A2A receptor (see Dunwiddie and Masino 2001 for review). To explore the possibility that A2 receptors contributed to either the cessation of firing and/or membrane hyperpolarization, the selective A2 receptor agonist CPCA (1 µM) was bath-applied to the slices. In contrast to the adenosine or A1-agonist-induced hyperpolarization, CPCA elicited a depolarization of 3.56 ± 0.65 mV (P < 0.001) as shown in Fig. 4, A and B. CPCA did not induce a termination in firing activity (n = 9) and a slight prolonging effect on action potential duration (+92 ± 49 µs, P < 0.05) was observed. Adenosine acts postsynaptically
Adenosine, acting at presynaptic A1 receptors, reduces the frequency of incoming postsynaptic currents (Oliet and Poulain 1999) (Fig. 5). However, bolus application of adenosine also causes a marked and lasting apparent outward current in magnocellular neurons (Fig. 5A). In horizontal slices of SON, there is an abundance of excitatory and inhibitory spontaneous postsynaptic activity (see Figs. 5 and 7). Although it is unlikely that presynaptic A1 receptors underlie changes in postsynaptic spike duration, it is possible that an adenosine-mediated reduction in presynaptic excitatory drive may reduce the firing frequency and ultimately lead to a postsynaptic hyperpolarization. It was therefore important to determine if adenosine was acting directly on postsynaptic receptors.
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-S (1–2 mM) to address this issue. GDP--S, an inactive analog of GDP, diffuses into the cell, outcompeting the endogenous GDP ultimately rendering G protein linked receptors ineffective. After waiting 10 min to allow the GDP--S to dialyze throughout the cell, adenosine was applied. In these experiments, GDP--S prevented not only the adenosine-mediated termination of firing activity, but also the A1 receptor-mediated reductions in spike duration and firing frequency (Fig. 6). Therefore the observed effects seem to be postsynaptic. Still, it could be argued that adenosine acting presynaptically could cause the release of another factor that could then bind a postsynaptic G protein–linked receptor, thereby inducing the observed effects. This, however, is unlikely because the effects are mediated by A1 receptors (Fig. 3), which reduce, not increase, the probability of transmitter release.
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), several of these cells showed marked increases in spontaneous EPSCs, as shown in Fig. 7, B1 and B2. The ability of endogenously generated adenosine to modulate magnocellular neuronal excitability was further tested using the adenosine uptake inhibitor dilazep bath-applied to the slices (1–100 µM). In all cases (n = 5), dilazep induced both a membrane hyperpolarization (–4.3 ± 1.8 mV) and termination of firing, suggesting a highly active adenosine uptake mechanism in this system (Fig. 7C). Co-localization of A1 receptors with supraoptic magnocellular neurons
These physiological and pharmacological data strongly suggest that there is a postsynaptic presence of adenosine A1 receptors in the SON and that they exert strong inhibitory effects on magnocellular excitability. Postsynaptic locations of A1 receptors were confirmed immunocytochemically on serial sections containing the SON, using polyclonal antibodies against the A1 receptor along with combined monoclonal antibodies against OT and VP. Although A1 receptor immunoreactivity in the SON has been described (Ikeda et al. 2001), the reports of functional A1 receptors on both astrocytes and pituicytes required a closer look at their localization with respect to magnocellular neurons (Abe and Saito 1998; Miyata et al. 1999; Rosso et al. 2002). In agreement with previous reports suggesting A1 localization on GABAergic and glutamatergic terminals, strong adenosine A1 receptor immunoreactivity was seen throughout the somatic area of the SON along with weaker labeling in the dendritic zone and the ventral glia lamina, as has been reported previously (Ikeda et al. 2001). Suggesting a postsynaptic locus, co-localization of A1 and OT/VP immunoreactivity was visible in virtually all SON-labeled cells (Fig. 8, A–C). Confocal z-series projections show immunoreactivity to two different adenosine A1 receptor antibodies in SON neurons (Fig. 8, D and E).
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Brief suprathreshold current pulses were given to cells to evoke single action potentials. Like the effects seen under conditions of spontaneous firing, application of adenosine or CPA caused a marked hyperpolarization (Fig. 9). The same current pulses were insufficient to evoke action potentials. Washout of adenosine returned the cells to firing in response to the current pulses. This further suggests that the changes in membrane potential are not due to changes in presynaptic activity.
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), application here of adenosine (100 µM) did not significantly alter this current (n = 5) in either amplitude or duration. However, in agreement with a report by Noguchi and Yamashita (2000), adenosine greatly reduced the duration of the Ca2+ spike (Fig. 10, A and B). To investigate the involvement of high-voltage Ca2+ channels, 100 µM adenosine was perifused in the presence of 100–200 µM Cd2+. Under theses conditions, an adenosine-induced suppression of firing activity persisted in five of six cases, suggesting the adenosinergic effect on membrane potential is not mediated through high-voltage Ca2+ channels.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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), inhibiting a variety of cell types in several brain regions (Chen and van den Pol 1997; Dolphin and Prestwich 1985; Fredholm et al. 1983; Lupica et al. 1992; Oliet and Poulain 1999; Paton 1981; Pittman 1999; Zhang and Schmidt 1999). This inhibition has largely been attributed to presynaptic mechanisms involving activation of A1 receptors (Cunha 2001; Fredholm and Dunwiddie 1988; Lupica et al. 1992), which can occur on both GABAergic and glutamatergic terminals. Adenosinergic control of SON neurons
Magnocellular neurons of the SON are part of an evolutionarily conserved system that monitors and responds to a variety of physiological conditions. Regulation of magnocellular neuronal excitability together with OT and VP release is influenced by several factors including numerous neurotransmitters (Hatton and Li 1998; Sladek and Kapoor 2001), surrounding glia (Hatton 1997; Hussy et al. 2001; Piet et al. 2002), osmolality of the external environment (Wakerley et al. 1978), and cell coupling via gap junctions (Cobbett and Hatton 1984). Unlike in vitro studies of most other brain areas, many cells of the SON remain spontaneously active in the slice, preserving activity similar to that observed in vivo and benefiting closer investigation of factors that modulate firing activity.
The adenosine A1 receptor is linked to the pertussis toxin (PTX)-sensitive Gi/o G proteins that, on activation, inhibit adenylyl cyclase, inhibit Ca2+ channels, activate phospholipase C, activate G protein–dependent inwardly rectifying K+ channels (GIRKs), and enhance the IAHP (Dunwiddie and Fredholm 1989; Dunwiddie and Masino 2001; Haas and Selbach 2000). Although a PTX-sensitive adenosinergic reduction of N-type Ca2+ channels has been reported (Barajas-Lopez et al. 1996; Mynlieff and Beam 1994; Park et al. 2001), the study by Noguchi and Yamashita (2000) found inhibition of N-type currents in dissociated SON cells to be insensitive to intracellularly applied PTX. Two possibilities may account for these differences: 1) the A1 receptor, like other receptors (Hawes et al. 2000; Luscher et al. 1997), can couple to more than one G protein in magnocellular neurons, thereby mediating its effects through multiple, including PTX-insensitive, pathways, and 2) the activated 
subunit is not subject to ADP-ribosylation by PTX. Interestingly, the report by Luscher et al. (1997) provided evidence of different G proteins coupled to pre- versus postsynaptic adenosine A1 receptors but did not address the possibility of differential G protein coupling specifically within the postsynaptic zone. Further studies monitoring distinct postsynaptic A1-mediated effects (e.g., hyperpolarization and either the reduction of spike duration or the reduction in Ca2+ influx) while inhibiting specific G proteins would address this issue. The presence of Gz, a PTX-insensitive member of the Gi/0 family, was immunocytochemically shown in supraoptic neurons (Noguchi and Yamashita 2000). However, it is thought that A1 receptors do not couple to this G protein (reviewed in Klinger et al. 2002).
Mechanisms of the adenosinergic reduction of spike duration
Our results show that adenosine has a strong influence, not only on the firing activity of SON neurons, but also on action potential duration. A possible explanation for the reduced duration is activation of postsynaptic GIRKs combined with a reduction of intracellular calcium. Evidence for the postsynaptic presence of GIRKs in the SON has been presented (Li et al. 2001a), and the A1 receptor can activate GIRKs pre- and postsynaptically (Wetherington and Lambert 2002). Because these cells are known to exhibit frequency-dependent spike broadening (Andrew and Dudek 1985; Bourque and Renaud 1985) and because the broadening is mediated by an increase in intracellular Ca2+, both a GIRK-induced reduction in frequency combined with a decreased Ca2+ flux through N-type channels could explain the adenosinergic effect on spike duration under conditions of spontaneous firing. A reduction of frequency-dependent spike broadening, however, was not a factor in the evoked action potential experiments shown in Fig. 9.
Alternatively, the reduction in action potential duration could be explained by an enhancement of the IAHP or IK. In support of this are studies showing the location and physiological activation of A1 receptors on axons (Swanson et al. 1995, 1998) along with earlier studies directly monitoring the effect of adenosine on the spike afterhyperpolarization (Haas and Greene 1984). However, the Ca2+ spike-generated IAHP seen in the presence of TTX and TEA was not enhanced by adenosine in either amplitude or duration. Therefore it seems possible that adenosine A1 receptors may act on IK or one of its underlying currents (e.g., the A-current) (Pan et al. 1994).
Inhibition of hormone release through A1 receptor activation?
Shortening action potential duration results in reduced transmitter release (Branchaw et al. 1998). Because SON neurons are peptidergic and peptidergic dense core vesicles (e.g., OT- or VP-containing) are not as readily exocytosed as small transmitter-containing clear secretory vesicles, a reduction in action potential duration would likely result in much-reduced levels of peptide release. Thus both the decreased firing and shorter action potential duration would presumably greatly reduce the amount of OT and VP released not only from the neural lobe, but also from dendrites within the nucleus itself. For a system vested with synthesizing and releasing peptide hormones that regulate parturition, lactation, diuresis, and natriuresis, this becomes important with respect to whole animal physiology. Under circumstances in which magnocellular A1 receptors are activated, it is possible that the clear, small, round vesicles and not the dense-core vesicles would be preferentially released.
Sources of adenosine
Our results are consistent with the presence of tonic endogenous A1 receptor activation, indicating that the neurons of the SON are kept in a continuous state of adenosine-induced inhibition. Although the exact source of this adenosine remains unknown, it is thought that most adenosine is probably not exocytosed. Rather, it is thought either to be transported into the extracellular environment via bi-directional nucleoside transporters or to result from the breakdown of ATP by endonucleases. Both the synaptic cleft and the neurohypophysis contain endonucleases (Dunwiddie et al. 1997; Wang et al. 2002) and large amounts of ATP are found inside synaptic vesicles—both dense-core and small, clear vesicles. The abundance of spontaneous activity seen in this system suggests that breakdown of ATP accounts for a significant source of available adenosine.
Furthermore, supraoptic neurons are known to be capable of dendritic release of peptides (Pow and Morris 1989). ATP contained in these vesicles could quickly get degraded to adenosine, which could act both pre- and postsynaptically to reduce activity in the SON. In support of this is recent evidence for an adenosinergic feedback from OT neurons onto GABAergic terminals (de Kock et al. 2003).
A2 receptors are not responsible for the inhibitory actions of adenosine
The A2 receptor-mediated depolarization of the cell's membrane potential suggests that adenosine in this system may induce opposing actions, depending on which receptors are activated. The CPCA-induced depolarization could result from either activation of presynaptic A2A receptors on glutamatergic terminals, thereby increasing glutamate release, or direct activation of postsynaptic A2 receptors. Although anatomical evidence for the presence of A2A receptors in the SON has been presented (Rosin et al. 1998), anatomical and physiological evidence regarding their precise localizations or hinting at their relative numbers is lacking. In addition to neurons, A2A receptors are expressed by astrocytes and endothelial cells (Fiebich et al. 1996; Li et al. 2001b; Schaddelee et al. 2003), two prominent cell types in the SON. If the A1 and A2 receptors are found localized to distinct, perhaps nonoverlapping cellular regions, afferent projections to those areas may selectively activate A1 versus A2 receptors. Activation of the pathway would cause an immediate afferent transmitter action (e.g., excitation by glutamate), followed by the purinergic effect. Further experiments monitoring an A2 effect on spontaneous and evoked PSCs and investigating potential postsynaptic effects are needed to address this issue.
That adenosine A1 receptor activation induces morphological changes in supraoptic and neurohypophasial astrocytes (Abe and Saito 1998; Miyata et al. 1999; Rosso et al. 2002), similar to those occurring during magnocellular system activation may seem paradoxical to the inhibitory effects on SON neuronal activity. However, this system is bestowed with a number of mechanisms functioning to maintain activity within an optimum range. An adenosinergic induction of astrocyte stellation resulting in reduced interneuronal space (thereby facilitating neuronal activation), while simultaneously reducing presynaptic transmitter release and potently inhibiting postsynaptic discharge, suggests adenosine's actions in this system to be complex and opens an important area for future investigation.
In this study, we determined that SON neurons contain functional postsynaptic adenosine A1 receptors that mediate a hyperpolarization concurrently with a reduction in spike duration. Adenosine acting at postsynaptic A1 receptors causes the cells to cease spontaneous firing activity, which would lead to much reduced peptide release from the neurohypophysis.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Address reprint requests and other correspondence to: T. A. Ponzio (E-mail: todd.ponzio{at}email.ucr.edu)
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REFERENCES |
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