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Calgary Brain Institute,
1 Neuroscience
2 Gastrointestinal Research Groups, Faculty of Medicine, University of Calgary, Calgary, Alberta, Canada T2N 4N1
3 Medical College of Wisconsin, Milwaukee, WI, USA
4 Departments of Anaesthesiology and Physiology and Biophysics, University of Washington, Seattle, WA, USA
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Abstract |
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(Received 13 April 2004;
accepted after revision 8 July 2004;
first published online 14 July 2004)
Corresponding author Q. J. Pittman: Neuroscience Research Group, Department of Physiology and Biophysics, Faculty of Medicine, University of Calgary, 3330 Hospital Drive NW, Calgary, AB, Canada T2N 4N1. Email: pittman{at}ucalgary.ca
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Introduction |
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In the supraoptic nucleus (SON), there is now good evidence that the neurohypophysial peptides arginine vasopressin (AVP) and oxytocin (OXT) are released from the somatodendritic compartment of magnocellular neurones (reviewed in Ludwig, 1995; Morris et al. 2000). A series of physiological and pharmacological studies have shown that OXT acts locally on OXT autoreceptors to elevate intracellular Ca2+ (Lambert et al. 1994; Ludwig et al. 2002). In keeping with this action, immunocytochemical studies have localized OXT receptors to OXT magnocellular neurones (Freund-Mercier et al. 1994; Adan et al. 1995). The locally released OXT both facilitates the activity of OXT magnocellular neurones and increases the axonal and dendritic release of the peptide (Moos et al. 1984; Neumann et al. 1996). In addition, we have shown that both exogenously applied and endogenously released OXT causes a reduction in glutamate-mediated excitatory postsynaptic currents (EPSCs) onto magnocellular neurones through an apparent action at a presynaptic terminal (Kombian et al. 1997). Similar results have been obtained for a presynaptic action of OXT upon GABAergic inhibitory postsynaptic currents (IPSCs) (de Kock et al. 2003). Given the known postsynaptic location of OXT receptors (Lambert et al. 1994; Freund-Mercier et al. 1994) we have tested the hypothesis that dendritically released OXT acts on the postsynaptic magnocellular neurone to indirectly inhibit glutamate release via an intermediary, retrograde messenger.
Recent studies, largely in hippocampus and cerebellum, but also in several other brain areas, have implicated endocannabinoids in retrograde signalling. Depolarization (Kreitzer & Regehr, 2001a,b; Ohno-Shosaku et al. 2001; Wilson & Nicoll, 2001; Trettel & Levine, 2003), an elevation in intracellular Ca2+ (Wilson & Nicoll, 2001; Brenowitz & Regehr, 2003), or activation of postsynaptic metabotropic receptors (Varma et al. 2001; Maejima et al. 2001; Di et al. 2003) cause the release of endocannabinoids that act at presynaptic terminals to inhibit either excitatory or inhibitory transmitter release through the activation of G-protein-coupled CB1 receptors.
There is indirect evidence for the action of cannabinoids on SON function. For over half a century it has been known that marihuana intake in man is associated with diuresis, suggestive of reduced AVP release (Tayleur Stockings, 1947; Ames, 1958), and a similar effect is seen in rats treated with the cannabinoid, 
9 tetra-hydro-cannabinol (9 THC) (Sofia & Barry, 1977). Similarly the administration of hashish (Frischknecht et al. 1980) or 9 THC (Borgen et al. 1971) to either lactating rats or mice is associated with reduced pup weight and a lack of milk transfer to the stomach of the pups, an effect consistent with reduced OXT release. In keeping with these observations, 9 THC has been reported to reduce the activity of the SON neurones, as indicated by an accumulation of neurosecretory material in their axons (Biswas & Ghosh, 1975). These early observations suggesting the inhibitory effects of cannabinoids in the SON prompted us to ask if they could alter synaptic transmission in the SON and also to determine whether they might be mediators of OXT action. In the present study, we show for the first time that endocannabinoids act as retrograde messengers in the SON and that CB1 receptors are localized on presynaptic terminals. In addition, we have found that the release and autocrine action of OXT subsequent to depolarization of SON magnocellular neurones plays an important role in inducing cannabinoid release.
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Methods |
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Slice preparation
Adult male Sprague-Dawley rats (100–150 g) were decapitated under deep halothane anaesthesia, the brain was removed and 250–300 µm thick coronal slices containing the SON were generated at 0–2°C in a low Ca2+, low Na+-containing buffer solution of composition (mM): NaCl (87), KCl (2.5), NaH2PO4 (1.25), MgCl2 (7), CaCl2 (0.5), NaHCO3 (25), glucose (25) and sucrose (20). Slices were incubated at 32–34°C for an hour and then at room temperature (22°C) in artificial cerebrospinal fluid (ACSF) of the following composition (mM): NaCl (126), KCl (2.5), NaH2PO4 (1.2), MgCl2 (1.2), CaCl2 (2.4), NaHCO3 (18) and glucose (11). Both solutions were continuously bubbled with a mixture of O2 (95%) and CO2 (5%).
Electrophysiological recording
ACSF-submerged slices were visualized by a DIC-IR microscope, and nystatin-perforated patch whole-cell recording (series/access resistance: 10–40 M
) was performed at 30–32°C using electrodes with a tip resistance of 3–7 M. The internal recording solution contained (mM): potassium gluconate (120), MgCl2 (5), EGTA (10) and Hepes (40). Nystatin was dissolved in DMSO with Pluronic F127 and added to the internal solution to yield a final concentration of 450 µg ml–1. The pH was adjusted to 7.3. All experiments were done on neurones voltage clamped at –80 mV using an Axopatch 1D amplifier and pCLAMP 9 software (Axon Instruments). Membrane currents were recorded without series resistance compensation, filtered at 1 kHz and digitized at 5–10 kHz and stored for off-line analysis. A 20 mV hyperpolarizing pulse lasting for 75 ms was applied regularly throughout each experiment, and the steady-state current and decay rate (
) of the capacitance transient were monitored as measures of input resistance and series/access resistance, respectively. Cells that showed more than 15% change in these parameters at the end of each experiment were excluded from further analysis. Magnocellular neurones were identified based on the delayed onset to action potential generation in response to positive current injection and all recordings in this paper were from magnocellular neurones. In some cells, AVP or OXT identity was ascertained in voltage clamp by current responses to hyperpolarizing voltage steps from –40 mV (Hirasawa et al. 2003a).
Synaptic currents (50–70% of maximal amplitude) were evoked with a bipolar stimulating electrode placed in the hypothalamic region dorsomedial to the SON, close to the optic tract. For all experiments, picrotoxin (50 µM) was added to ACSF to block GABAA receptor-mediated chloride currents and give pharmacologically isolated EPSCs. EPSCs were mediated by non-NMDA receptors as they were sensitive to 10 µM DNQX, a non-NMDA receptor antagonist.
To monitor paired pulse ratio (PPR), a pair of EPSCs was evoked with an interval of 50 ms and the ratio was calculated as EPSC2/EPSC1. To isolate miniature EPSCs (mEPSCs), tetrodotoxin (1 µM) was also added to the ACSF to eliminate action potential-driven EPSCs. Previously it has been shown that in coronal SON slice preparation, virtually all spontaneous events are action potential independent (Kombian et al. 2000), enabling us to monitor miniature events and evoked response concurrently, by omitting tetrodotoxin in some of the experiments. The results obtained with or without tetrodotoxin were not significantly different, thus the data were combined.
Control data were collected for 3–5 min prior to drug application. Baseline was calculated as the mean of the values obtained during the control period. mEPSCs were detected using MiniAnalysis software (Synaptosoft Inc.) and counted if amplitude was larger than 4–5 times the RMS noise with fast rise times (0.4–3 ms measured 10–90% from baseline to peak) and exponential decay. All values are expressed as mean ± S.E.M. Statistical comparisons were performed by using appropriate tests, i.e. Student's paired and unpaired t tests, ANOVA and the Kolmogorov-Smirnov test. P < 0.05 was considered significant.
All substances were prepared as x 1000 stock solutions, and diluted to their final concentration in ACSF just before use. The source of all chemicals and drugs was Sigma except for the following: oxytocin and Manning compound (Bachem), WIN55,212-2 and AM251 (Tocris Cookson), tetrodotoxin (Alomone Laboratories).
Immunohistochemistry
Rats (n = 4) were deeply anaesthetized with pentobarbital sodium (65 mg kg–1 I.P.; Somnotol, MTC Pharmaceuticals, Cambridge, ON, Canada), and fixed by intracardiac perfusion with 100 ml physiological saline followed by 500 ml 4% paraformaldehyde (pH 7.3). Brains were removed and fixed overnight in 4% paraformaldehyde at 4°C. They were then transferred to 20% sucrose in 0.1 M phosphate buffer at 4°C overnight. Coronal sections of brain at levels containing the SON were cut at 50 µm and floating sections were washed for 3 x 10 min in phosphate-buffered saline (PBS) containing 0.1% Triton X-100 and then incubated in a rabbit anti-CB1 receptor antibody (1: 1000) (Tsou et al. 1998a; Ong & Mackie, 1999; Van Sickle et al. 2003) or a rabbit anti-fatty acid amide hydrolase antibody (FAAH, 1: 200) (Tsou et al. 1998b) at 4°C for 48 h. Specificity was confirmed by preincubation of the antibodies (24 h at 4°C) with the peptides used to raise the antibodies (10 nM in diluted antibody). This procedure completely abolished labelling in all cases. Sections were washed with 0.1% Triton X-100 in PBS and incubated for 1–2 h at room temperature in the secondary antisera (donkey anti-rabbit conjugated to CY3; 1: 100, Biocan Scientific, Mississauga, ON, Canada) followed by washing (3 x 10 min in PBS) and mounting in bicarbonate-buffered glycerol (pH 8.6).
Confocal microscopy
Samples were viewed on an Olympus Fluoview FV300 microscope system using krypton–argon and helium–neon lasers. Differential visualization of the fluorophores FITC (excitation 490 nm and emission 520 nm), and CY3 (excitation 552 nm and emission 565 nm) was accomplished through the use of specific filter combinations. Samples were scanned sequentially if double-labelled, to avoid any potential for bleed-through of fluorophores. Images of 1024 pixels x 1024 pixels were obtained under identical exposure conditions (pinhole aperture, laser strength, scan speed, Kalman averaging x 2) and were processed identically using Fluoview software and then CorelDraw 11. Confocal micrographs are digital composites of Z-stack scans through 1 µm optical sections, as described in the figure legends.
Electron microscopy
Rats (n = 3) were deeply anaesthetized with halothane and perfused through the heart with a mixture of 4% paraformaldehyde and 0.2% glutaraldehyde in 0.1 M PBS, pH 7.4. Brains were then removed and postfixed overnight in 4% paraformaldehyde at room temperature and incubated in 14% glycerol and 30% sucrose in PBS until further processing. Frozen sections (40 µm) were first preincubated for 15 min in 1% sodium borohydride in PBS and then in a blocking buffer containing 0.1% Tween 20 and 5% donkey serum in PBS. Primary antibodies against CB1 receptors (as above) were used at 1: 500 dilution in PBS + 0.1% Tween 20 and incubated for 48 h at 4°C. After extensive washing with PBS, the slices were incubated with a 1/500 dilution of a biotinylated goat anti-rabbit IgG (Santa Cruz) for 90 min at room temperature and then for 45 min in a 1/500 dilution of the Vectastain ABC kit (Vector Laboratories) followed by DAB (Vector Laboratories) for 8 min at room temperature. Tissues were then postfixed with 2.5% glutaraldehyde in PBS for 30 min, then with 1% osmium tetroxide in PBS for 1 h at 4°C and incubated in 70% ethanol overnight at 4°C. Dehydration was performed in graded alcohol and then after propylene oxide treatment the tissues were embedded in a mixture of Araldite and Epon resins. Silver ultrathin sections were obtained using a Reichert ultramicrotome and collected on copper grids. Pictures were taken with a digital camera (AMT) mounted on a Hitachi H 7500 electron microscope.
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Results |
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The role of endocannabinoids in synaptic excitation of magnocellular neurones was investigated by testing the effect of a cannabinoid receptor agonist, WIN55,212-2 (1 µM) on evoked EPSCs in SON. WIN55,212-2 induced a significant, long-lasting, partially reversible inhibition of EPSCs to 50.6 ± 9.5% of control (n = 8, Fig. 1A and C). Included in this population were both AVP and OXT cells, identified by standard electrophysiological approaches. This effect was prevented by preincubation with a CB1 receptor antagonist, AM251 (1 µM; 97.0 ± 5.1% of control, P > 0.05, n = 5; Fig. 1B and C). AM251 alone had a highly variable, but not significant, effect on the EPSC amplitude (146.1 ± 22.5% of control, P > 0.05, n = 7).
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Spontaneous presynaptic release of transmitter can be influenced by intraterminal Ca2+ level, which can fluctuate due to the influx or release of Ca2+ from internal stores. Alternatively, spontaneous release can be modulated by mechanism(s) downstream of intraterminal Ca2+ level. To differentiate amongst these possibilities, one can utilize agents that directly facilitate the release process downstream of Ca2+ entry or release and determine how CB1 receptor activation influences transmitter release activated by such agents. We used nifedipine, a secretagogue that induces spontaneous glutamate release independently of Ca2+ influx or release from the internal stores (Hirasawa & Pittman, 2003b). Nifedipine (10 µM) induced a 14.1(± 3.2)-fold increase in mEPSC frequency (n = 5). WIN55,212-2 inhibited nifedipine-induced mEPSC frequency to 40.1 ± 5.0% of the nifedipine effect (P < 0.05, n = 5; Fig. 3B and C) but was without effect on the amplitude of mEPSCs (99.9 ± 6.4% of nifedipine effect, P > 0.05, n = 5; Fig. 3B and D). Thus, WIN55,212-2 seems to be able to modulate a release process independently of Ca2+ influx or release from intracellular stores.
Cannabinoid mediation of oxytocin action
The inhibitory effect on evoked EPSCs by CB1 receptor activation (Fig. 1) was similar to the previously reported effect of OXT on SON excitatory transmission. Thus we tested the hypothesis that OXT exerts its effect through endocannabinoids. The effect of OXT on evoked EPSCs was tested in the presence of a CB1 receptor antagonist. While OXT (1 µM) alone induced a significant decrease in EPSC amplitude (65.2 ± 2.5% of control, P < 0.05, n = 14; Fig. 4A and D), the effect was blocked by pretreatment with AM251 (103.7 ± 3.5% of AM251 alone, P > 0.05, n = 6; Fig. 4B and D). Furthermore, the OXT effect was occluded in the presence of WIN55,212-2 (96.8 ± 4.5% of WIN55,212-2 alone, P > 0.05, n = 3; Fig. 4C and D). These results indicate that OXT inhibits EPSCs through the activation of CB1 receptors, by the release of endocannabinoids.
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In addition to the effect on evoked EPSCs, depolarization induced a reduction in the frequency of mEPSCs (71.6 ± 4.4% of control, P < 0.05, n = 8, Fig. 7D), without affecting their amplitude (98.7 ± 6.8% of control, P > 0.05, n = 8). This result is similar to the effect of WIN55,212-2 on mEPSCs (see Fig. 3).
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Immunohistochemical localization of the cannabinoid degradative enzyme FAAH is depicted in Fig. 8A1. Magnocellular cell bodies displayed immunoreactivity for the enzyme. In contrast, CB1 receptor immunoreactivity appears to be an inverse of FAAH localization (Fig. 8A2). At electron microscopic level, the presence of CB1 receptor immunoreactivity was clearly localized to axon terminals forming asymmetric or symmetric synapses on magnocellular dendrites (Fig. 8B–F). However, immunoreactive product was not found in postsynaptic structures.
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Discussion |
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Presynaptic action of cannabinoids on excitatory transmission
Regulation of magnocellular neuronal activity is accomplished not only through the modulation of intrinsic conductances (Bourque et al. 1993), but also through excitatory and inhibitory afferent inputs. Glutamatergic inputs comprise the majority of excitatory inputs onto these cells (Theodosis, 1988; Van den Pol et al. 1990) and presynaptic receptors on these terminals are now recognized to be major loci for the control of SON (Pittman, 1999). Di et al. (2003) have previously reported in paraventricular nucleus that the presynaptic effects of glucocorticoids are mediated by the release of endocannabinoids from postsynaptic cells. Our demonstration that WIN55,212-2 reduces both evoked and mEPSCs indicates that cannabinoid receptors can also modulate excitatory glutamatergic inputs in the SON. Both PPR and mEPSC frequency, but not amplitude, are altered, indicating that the presynaptic terminal is the target of modulation. These effects are most likely mediated by CB1 receptors, because the WIN55,212-2 effect is blocked by AM251, a CB1 receptor antagonist, which is consistent with CB1 receptors being the major cannabinoid receptors in the central nervous system (Onaivi et al. 2002). In order to localize CB1 receptors, immunohistochemical studies were performed with an antibody that was highly specific for CB1 receptors. The immunoreactive material at the light microscopic level appeared to surround the magnocellular neurones. At the electron microscopic level, this immunoreactivity was clearly localized to axon terminals that form asymmetrical or symmetrical synapses with dendrites of magnocellular neurones, which indicates that CB1 receptors are expressed at both the excitatory and inhibitory presynaptic terminals in the SON.
The mechanism by which CB1 receptor activation reduces glutamatergic EPSCs was investigated with the use of the secretagogue, nifedepine, that is thought to directly target the release process (Hirasawa & Pittman, 2003b). The observation that CB1 receptor activation was effective in reducing nifedepine-stimulated glutamate secretion suggests an action of cannabinoids at the release process. As nifedepine-stimulated mEPSCs are insensitive to Ca2+ channel blockers or to internal Ca2+ depletion (Hirasawa & Pittman, 2003b), this would indicate that the effect can occur downstream from Ca2+ entry. Similar results have been reported elsewhere (Diana et al. 2002; Azad et al. 2003). However, there is also evidence that cannabinoids can inhibit voltage-dependent Ca2+ and K+ conductances (Mackie et al. 1995; Twitchell et al. 1997; Sullivan, 1999; Kreitzer & Regehr, 2001b; Diana et al. 2002). Indeed, the OXT effect in the SON can be occluded by N- and P/Q-type Ca2+ channel blockers (Hirasawa et al. 2001) suggesting that CB1 receptors may also modulate these Ca2+ channels in the SON.
There have been reports that some CB1 receptor antagonists, including AM251, can act as inverse agonists when CB1 receptors are overexpressed in transfected cell lines (MacLennan et al. 1998; New & Wong, 2003). However, in our study, the antagonist (at an effective blocking concentration of 1 µM) was without significant effect upon evoked EPSCs, thereby arguing against this possibility. Nonetheless, despite the lack of a significant effect overall, in some cells AM251 did clearly increase the evoked EPSC, as is indicated by the large variation in the response (146.1 ± 22.5% of control). Thus, it is possible that there is ongoing endocannabinoid activity at the glutamatergic synapse in the SON slice or that, in some synapses, AM251 may have inverse agonist activity.
Endocannabinoids mediate oxytocin action on excitatory transmission
The present demonstration that the OXT-induced reduction in evoked EPSC amplitude is antagonized by the CB1 receptor antagonist (and occluded by a cannabinoid agonist) could be accounted for through several mechanisms. One possibility is that the CB1 receptor antagonist is capable of blocking a presynaptic OXT receptor. We believe this to be unlikely; the CB1 receptor (Matsuda et al. 1990) and the OXT receptor (Kimura et al. 1992) have both been cloned and although both are G-protein coupled, they show little homology. Furthermore, the structural basis of ligand binding for neurohypophysial receptors has been well described (Barberis et al. 1998) and it appears highly unlikely that the diarylpyrazole, AM251, that is structurally very different from the neurohypophysial peptides, would interfere with OXT binding (admittedly, this does not negate the possibility that AM251 could interfere with OXT binding through a non-competetive, allosteric interaction). A second possibility is that OXT and CB1 receptors coexist on the same terminal and that second messenger systems downstream to either of the receptors somehow interact with each other. For example, it has been reported in transfected neurones that the CB1 receptor can sequester Gi/o proteins (Vasquez & Lewis, 1999); such sequestration would make them unavailable to other signalling pathways such as those required for OXT signalling. In support of this possibility, a cannabinoid agonist WIN55,212-2 and OXT occluded each other. However, if this were the case, one would expect that the AM251, a CB1 receptor antagonist, would be ineffective against OXT action, but as we have seen, it blocked the OXT effect. A third possibility is a sensitization of OXT receptors by active CB1 receptors, similar to the interaction observed between orexin and CB1 receptors (Hilairet et al. 2003). However, the absence of tonic endocannabinoid tone, which is apparent as a lack of AM251 effect, argues against such a mechanism. Also, WIN55,212-2 should facilitate OXT effect rather than occlude it, if such a mechanism exists. The most likely explanation for our findings is that exogenously applied OXT acts at postsynaptic OXT autoreceptors to release endocannabinoids that then act on the presynaptic terminal to inhibit glutamate release. Consistent with this idea is the fact that activation of postsynaptic OXT receptors has been shown to elevate intracellular Ca2+ (Lambert et al. 1994), a prerequisite for endocannabinoid synthesis and release in many neurones (Wilson & Nicoll, 2001; Brenowitz & Regehr, 2003). As we have demonstrated the presence of FAAH in SON neurones, and the presence of the enzyme is indicative of endocannabinoid signalling (Tsou et al. 1998b; Egertova et al. 1998), this is additional evidence that magnocellular neurones in the SON release endocannabinoids. Thus OXT is one of several metabotropic transmitters that have now been shown to activate endocannabinoid signalling. It is possible also that the activation of other metabotropic receptors on magnocellular neurones could cause endocannabinoid synthesis and release in a manner similar to the one described here for OXT.
Comparison of WIN55,212-2 and OXT action
If OXT action is mediated by endocannabinoids, there ought to be identity of action of OXT and WIN55, 212-2. With respect to the evoked EPSC, this appears to be the case (Kombian et al. 1997; Hirasawa et al. 2001); however, as for mEPSCs, OXT was relatively ineffective (Hirasawa et al. 2001) while WIN55,212-2 inhibited mEPSCs. The present study indicates that this discrepancy derives from mixed excitatory and inhibitory effects of OXT. The inhibitory effect seems to be mediated by CB1 receptors, since it was blocked by the CB1 antagonist. The mechanism of the excitatory effect of OXT in the presence of AM251 is unknown: possibilities include the direct action of OXT on presynaptic terminals inducing Ca2+ release from internal stores, leading to increased spontaneous glutamate release, or involvement of yet unknown excitatory retrograde messengers. It is curious that, whereas mEPSC frequency increased in the presence of AM251, there was no consistent increase in the size of the evoked EPSCs. However, in previous studies we have also observed large alterations in mEPSC frequency without an accompanying change in the size of evoked EPSCs (Kombian et al. 1997; Hirasawa et al. 2001). The discrepancy might arise from different Ca2+ sensitivity for evoked versus spontaneous transmitter release (Ravin et al. 1997).
Another factor that might account for the difference is the postsynaptic effect of OXT. Our previous study has demonstrated modulatory effects of neuropeptides on AMPA-induced postsynaptic currents in both OXT and AVP neurones (Hirasawa et al. 2003a), which would have little influence on the mEPSC frequency but would affect the amplitudes of evoked and miniature EPSCs. However, in the presence of AM251, OXT had no effect on these parameters, i.e. evoked or miniature EPSC amplitudes (Figs 4 and 5). This discrepancy could be due to differential effects of neuropeptides on AVP and OXT neurones (inhibitory effect mediated by V1a receptors and excitatory effect mediated by OXT receptors, respectively), because in the current study we recorded from a mixture of both population of neurones. This could be the case, as OXT may have significant action not only at OXT receptors but also at V1a receptors (Chen et al. 1999). Indeed, the majority of AVP neurones (identified by their electrophysiological fingerprints) showed a decrease in mEPSC amplitude in response to OXT application while most OXT neurones responded with an increase in mEPSC amplitude, which is in agreement with our previous findings.
We still stand by our previous conclusion that OXT inhibits presynaptic Ca2+ channels, possibly via endocannabinoids, because of the fact that the OXT effect was partially occluded by Ca2+ channel blockers (Hirasawa et al. 2001). Future investigation will be required to elucidate whether Ca2+ channel blockers also occlude the cannabinoid effect in a manner similar to OXT. There remains an anomaly in the effects of exogenous OXT and endogenous neuropeptide released by postsynaptic depolarization, i.e. exogenous OXT induced diverse effects on mEPSCs (Fig. 5) whereas endogenously released neuropeptide seemed to induce only inhibitory effects on mEPSCs (Fig. 7). One possible explanation for this discrepancy is that bath-applied OXT may, at the doses we used, have effects that do not exactly mimic those seen with endogenously released OXT that would have a much more localized action.
We have confirmed an action of OXT on both OXT and AVP cells identified by electrophysiological fingerprint in the SON (data not shown) and, in the present study, WIN55,212-2 was effective in every cell tested, whether they showed an OXT or an AVP electrophysiological fingerprint. Thus it is possible that OXT action on OXT cells releases sufficient endocannabinoids to diffuse to neighbouring synapses on both OXT and AVP cells. An alternative possibility is that OXT action at V1a receptors on AVP neurones also releases endocannabinoids. Although the present set of experiments did not address the potential role of AVP in activating endocannabinoid release, the fact that it also elevates intracellular Ca2+ (Gouzenes et al. 1999) makes it likely that it could do so in a manner similar to the one we have described for OXT. Indeed Manning compound is effective in blocking both V1a and OXT receptors, leaving the possibility that the retrograde inhibition is also induced by AVP.
The cooperative action of OXT and endocannabinoids and its functional implications
We were able to elicit a short-lasting inhibition of evoked and miniature EPSCs by depolarization of the postsynaptic magnocellular neurone, as we have previously reported (Kombian et al. 1997). We also confirmed that this depolarization-induced inhibition could be blocked by Manning compound, an OXT/V1a receptor antagonist (Kombian et al. 1997). In addition, the present study shows that the same inhibition induced by postsynaptic depolarization can be prevented in the presence of a CB1 receptor antagonist, thereby providing evidence for endocannabinoid release. Our first thought was that the OXT/V1a receptor antagonist was non-specifically blocking the CB1 receptor on the presynaptic terminal. However, this was not the case, as it did not block WIN55,212-2 action. Thus the similar blockade by both Manning compound and AM251 suggests that endogenous release of both endocannabinoid and the neurohypophysial peptides are required to inhibit afferent synapses. The most plausible explanation for how this might happen is that depolarization of the magnocellular neurone in itself is insufficient to cause enough endocannabinoid synthesis and release to inhibit the evoked EPSC in the slice. Rather, as depicted in Fig. 9, the depolarization-induced dendritic release of OXT needs to activate local autoreceptors on the same neurone, which in turn causes the synthesis and release of sufficient endocannabinoids to diffuse to the presynaptic terminals and inhibit the release of glutamate. Whether the endocannabinoid synthesis involves Ca2+ release from the internal store induced by OXT receptor activation (Lambert et al. 1994) remains to be elucidated. Meanwhile, Ca2+ release induced by activation of the OXT receptor will further stimulate OXT release creating a feed-forward activation of the system (Moos et al. 1984; Ludwig et al. 2002), which may underlie the prolonged inhibition caused by postsynaptic depolarization (order of minutes). In addition, OXT will also activate receptors (OXT receptors and possibly V1a receptors) expressed on neighbouring cells and induce endocannabinoid signalling at their synapse. Thus, dendritically released OXT may initiate a temporal and spatial spread or expansion of endocannabinoid signalling in the nucleus. Diffusion of endocannabinoids seems to be limited to individual synapses in the paraventricular nucleus; Di et al. (2003) have shown that blockade of G-protein signalling in the postsynaptic neurone prevents endocannabinoid release induced by glucocorticoids and the resulting synaptic inhibition. To understand whether endocannabinoids can diffuse to neighbouring synapses to modulate neurotransmission in the SON will require future investigation.
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References |
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