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NEUROSCIENCE |
1 Department of Anesthesiology, University of Washington, Seattle, WA 98195, USA
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Abstract |
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9-tetrahydrocannabinol, which has been shown to attenuate DSE, antagonizes MSE. Finally, we have distinguished a neuronal subpopulation that exhibits DSE and a differential complement of MSE-mediating Gq-coupled receptors, making possible contrasting studies of MSE. Autaptic endocannabinoid signalling is rich, robust and complex in a deceptively simple package, including a previously unreported postsynaptic mechanism of adaptation in addition to known presynaptic CB1 desensitization. These adaptive sites offer novel targets for modulation of endogenous cannabinoid signalling.
(Received 19 July 2006;
accepted after revision 14 November 2006;
first published online 16 November 2006)
Corresponding author A. Straiker: Department of Anaesthesiology, University of Washington, Seattle, WA 98195, USA. Email: straiker{at}u.washington.edu
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Introduction |
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In cell populations that exhibit DSI or DSE, a parallel system of retrograde inhibition has often been observed. This retrograde inhibition uses the same presynaptic receptors and messengers (CB1 and endocannabinoids) as DSI/DSE but is distinct insofar as it is activated not by postsynaptic depolarization but rather via Gq-coupled metabotropic muscarinic (M1/M3) and/or metabotropic glutamate (mGluR) (group I) receptors (Maejima et al. 2001; Kim et al. 2002). These receptors activate phospholipase C, cleaving phosphatidylinositol 4.5-bisphosphate (PIP2) into inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG), the latter being one precursor of 2-AG, a candidate endocannabinoid. Additionally, release of intracellular calcium by IP3 may augment endocannabinoid production. We will refer to this system of retrograde inhibition as metabotropic suppression of excitation (MSE) and inhibition (MSI).
We have recently reported the presence of robust DSE in excitatory autaptic hippocampal neurons (Straiker & Mackie, 2005), which served as a simple model for the high resolution study of DSE. We have now determined that MSE is also present in this model system. MSI has been reported in the hippocampus while MSE has been described in other brain regions but not in the hippocampus (Maejima et al. 2001; Hirasawa et al. 2004; Kushmerick et al. 2004). Therefore hippocampal MSE remains entirely unexplored. Is hippocampal MSE similar to MSI? Does it exhibit synergy with DSE? What is the identity of the endocannabinoid-mediating MSE? How does the principal psychoactive constituent of cannabis, 9-tetrahydrocannabinol (9-THC), interact with this form of cannabinoid-dependent retrograde signalling? Does MSE desensitize? We have used the autaptic preparation to explore these and other questions.
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Methods |
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All procedures used in this study were approved by the Animal Care Committee of the University of Washington and conform to the Guidelines of the National Institutes of Health on the Care and Use of Animals. Mouse (CD1 strain) hippocampal neurons isolated from the CA1–CA3 region were cultured on micro-islands as previously described (Furshpan et al. 1976; Bekkers & Stevens, 1991). Neurons were obtained from animals (at postnatal day 0–2, killed via rapid decapitation without anaesthesia) and plated onto a feeder layer of hippocampal astrocytes that had been laid down previously (Levison & McCarthy, 1991). Cultures were grown in high-glucose (20 mM) type medium containing 10% horse serum, without mitotic inhibitors and used for recordings after 8 days in culture and for no more than 3 h after removal from culture medium (Straiker & Mackie, 2005). All drugs were tested on cells from at least two different preparations.
Electrophysiology
When a single neuron is grown on a small island of permissive substrate, it forms synapses – or ‘autapses’
– onto itself. All experiments were performed on isolated autaptic neurons. Whole-cell, voltage-clamp recordings from autaptic neurons were carried out at room temperature using an Axopatch 200A amplifier (Axon Instruments, Burlingame, CA, USA). The extracellular solution contained (mM): NaCl 119, KCl 5, CaCl2 2.5, MgCl2 1.5, glucose 30 and Hepes 20, and 3 µM bovine serum albumin (as a carrier for the lipophilic cannabinoids). Continuous flow of solution through the bath chamber (
2 ml min–1) ensured rapid drug application and clearance. Drugs were typically prepared as a stock, then diluted into extracellular solution at their final concentration and used on the same day. In general, positive results were coupled on the same day with negative controls. Conversely, negative results for a given drug (e.g. in knockout cultures) were coupled on the same day with positive controls for that drug in control cells.
Recording pipettes of 1.8–4 M
were filled with solution containing (mM): potassium gluconate 121.5, KCl 17.5, NaCl 9, MgCl2 1, Hepes 10, EGTA 0.2, MgATP 2 and LiGTP 0.5. Access resistance was monitored and only cells with a stable access resistance were included for data analysis.
Conventional stimulus protocol. The membrane potential was held at –70 mV and EPSCs were evoked every 20 s by triggering an unclamped action current with a 1.0 ms depolarizing step. The resultant evoked waveform consisted of a brief stimulus artifact (i.e a large downward spike representing inward sodium currents) followed by the slower EPSC. The size of the recorded EPSCs was calculated by integrating the evoked current to yield a charge value (in pC). Calculating the charge value in this manner yields an indirect measure of the amount of neurotransmitter released while minimizing the effects of cable distortion on currents generated far from the site of the recording electrode (the soma). Data were acquired at a sampling rate of 5 kHz.
DSE stimuli. After establishing a 10–20 s baseline consisting of 1 ms depolarizations at 0.5 Hz, DSE was evoked by depolarizing to 0 mV for 1–10 s, followed by resumption of a 0.5 Hz stimulus protocol for 10 to > 80 s, as necessary.
Effects of MSE on paired-pulse responses were studied by paired trials in which two pulses (60 ms interval) were applied prior to and after an MSE-evoking treatment. The amplitude ratios of the pre-MSE pairs and the post-MSE pairs were averaged and compared via paired Student's t test.
Inhibitory neurons were identified and excluded based on characteristically slow IPSC time courses. When the identity of the neuron was in doubt, the AMPA receptor inhibitor 6-cyano-7-nitroquinoxaline-2.3-dione (CNQX) was applied to verify the presence of excitatory currents.
Identification of Type O neurons.
We noted that in some excitatory neurons, oxotremorine-M (oxo-M) inhibited EPSCs independently of CB1. We further observed that these neurons shared a constellation of physiological and pharmacological characteristics sufficient to conclude that we were dealing with a discrete and identifiable neuronal subpopulation, which we refer to as Type other or ‘O’. The neurons were not morphologically distinct, but exhibited a specific complex (often explicitly double-peaked) EPSC. EPSCs in Type O neurons were also uniquely inhibited by the 
1-adrenergic agonist phenylephrine (PE, 10–50 µM) and the 5-HT1 receptor agonist 5-carboxamidotryptamine (5-CT) (10 µM). Taken together, these criteria allowed identification of these neurons with considerable confidence.
Drugs
Drugs were purchased from Tocris Cookson (Ellisville, MO, USA), Cayman Chemical (Ann Arbor, MI, USA) or Sigma-Aldrich (St Louis, MO, USA). Heterozygote (CB1+/–) mice to establish our colony were generously provided by Dr Catherine Ledent (University of Brussels, Belgium; Reibaud et al. 1999).
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Results |
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Hippocampal MSI is a well-established phenomenon in both culture and slice preparations, occurring via Gq-coupled muscarinic receptors and mGluRs (Group I) (Kim et al. 2002; Ohno-Shosaku et al. 2002a, 2003; Hashimotodani et al. 2005). MSE has been reported in several regions of the brain, but not in hippocampus (Maejima et al. 2001; Hirasawa et al. 2004; Kushmerick et al. 2004). In order to explore a potential retrograde inhibitory capability for Gq-coupled receptors in CNQX-sensitive excitatory cultured hippocampal neurons, we examined muscarinic and mGluR agonists.
Muscarinic receptors mediate MSE in autaptic hippocampal neurons
We first tested the muscarinic agonist oxo-M in the dominant population of excitatory hippocampal neurons. Oxo-M readily inhibits EPSCs in these neurons (10 µM, 0.51 ± 0.09 of control, n = 7, Fig. 1A and B). Oxo-M-induced inhibition of EPSCs is blocked by the CB1 antagonist SR141716 (100 nM, 1.09 ± 0.09 of control, n = 8), and is absent in neurons cultured from CB1 knockout (CB1–/–) mice (1.22 ± 0.08 of control, n = 14), indicating that it requires presynaptic CB1 receptor activation (Fig. 1A and B). The recovery time for oxo-M inhibition (Fig. 1A; half life decay time (t1/2) = 38 s) is similar to that for DSE. The fact that oxo-M often potentiated EPSCs in the absence of CB1 receptors suggests that oxo-M has an additional action in these neurons.
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MSE appears to develop alongside DSE (i.e. one was rarely observed without the other) as cultures mature and, as with DSE, the extent of inhibition by MSE does not increase with age of neurons in culture (data not shown). Furthermore, the maximal magnitude of oxo-M-induced MSE is generally somewhat less than that of DSE in the same cell (oxo-M, 0.64 ± 0.05; DSE (same cell), 0.51 ± 0.05 of control, n = 22; Fig. 1D). Two brief applications of oxo-M produced the same degree of MSE, highlighting the stability of the response (1st treatment, 0.62 ± 0.09; 2nd treatment, 0.60 ± 0.07, n = 7; Fig. 1E).
The calcium requirements of endocannabinoid signalling have recently been revisited (Hashimotodani et al. 2005; Isokawa & Alger, 2006). Early evidence suggested that the principal source of calcium for DSE derives from external calcium entering neurons via voltage-sensitive calcium channels (Lenz et al. 1998). MSE on the other hand appeared to rely chiefly on calcium released from postsynaptic calcium stores (Robbe et al. 2002). Melis et al. (2004a) reported that 2-AG-mediated suppression of inhibition in the ventral tegmental area was dependent on calcium stores and Isokawa & Alger (2006) have reported similar findings for hippocampal DSI (with a greater role in younger animals). We have recently found that the Ca2+ ATPase inhibitor thapsigargin, which depletes calcium stores, at least partly suppresses DSE in autaptic hippocampal neurons (Straiker & Mackie, 2005). All of these results suggest a role for calcium derived from intracellular stores in eliciting DSE/DSI. Here we tested the effect of thapsigargin on MSE, and found MSE to be diminished after exhaustion of the thapsigargin-sensitive calcium stores (Fig. 1F; thapsigargin (1 µM, 20+ min) + oxo-M, 0.84 ± 0.06; n = 14; P < 0.0001 versus oxo-M control (see above), Student's t test). Our findings indicate that MSE relies at least in part on intracellular calcium stores.
MSE is thought to occur following Gq-coupled receptor activation of PLC and the production of DAG, the precursor of 2-AG, culminating in the postsynaptic production of endocannabinoids. We tested this hypothesis by treating neurons with the PLC inhibitor U-73122 (Fig. 1G; 3 µM, 2.5–5 min; same-cell experiments: oxo-M control, 0.56 ± 0.03, oxo-M + U73122, 0.91 ± 0.07, n = 4, P < 0.05 by paired t test). Thus, PLC appears to be an obligatory participant in MSE.
As mentioned above, two brief applications of oxo-M resulted in comparable inhibition; however, repeated/extended applications of oxo-M induce a desensitization of MSE (Fig. 2A; 1st oxo-M treatment, 0.65 ± 0.03; 2nd oxo-M treatment (same cell) after desensitization, 0.91 ± 0.04, n = 7; P < 0.005 paired t test). Notably, this desensitization occurs independently of DSE (i.e. DSE remains robust in the same neurons even as MSE desensitizes; Fig. 2A and B; DSE inhibition before desensitization, 0.41 ± 0.07; after desensitization, 0.49 ± 0.07, n = 6, P > 0.05 paired t test), suggesting that MSE desensitization is not due to downregulation of CB1 signalling. It is likely that it is due to downregulation of one or more components involved in postsynaptic endocannabinoid production, which is not shared with endocannabinoid production during DSE. Consequently, in subsequent experiments that called for multiple applications of MSE agonists, only brief applications were used to avoid induction of desensitization.
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5 µM). In same-cell experiments, we found that gallamine attenuated oxo-M-induced (2 µM) inhibition of EPSCs (Fig. 3D, control oxo-M inhibition, 0.51 ± 0.10; oxo-M plus gallamine, 0.77 ± 0.14, n
= 5, P < 0.01 by paired t test). Our results suggest that Type O neurons express functional presynaptic M2 muscarinic receptors capable of modulating glutamate release.
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We additionally tested (S)-3,5-dihydroxyphenylglycine (DHPG), a selective agonist for the Gq-coupled Group I mGluRs. DHPG (100 µM) also inhibited EPSCs (Fig. 4A and B; 0.74 ± 0.08, n = 10), though not in CB1–/– cultures (Fig. 4B; 0.99 ± 0.05, n = 3) or in the presence of SR141716 (Fig. 4B; 1.05 ± 0.07, n = 5). DHPG increased the paired-pulse ratio indicating a presynaptic site of action (Fig. 4C; paired-pulse ratio: 1st pair, 0.78 ± 0.04; 2nd pair, 0.85 ± 0.04, n = 6, P < 0.05 by paired t test). Thus in addition to a muscarinic system mediating retrograde inhibition of excitation via CB1 receptors, autaptic hippocampal neurons also support MSE via Gq-coupled group I mGluR activation.
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Agonists for other Gq-coupled receptors fail to elicit MSE
Given that the two postsynaptic Gq-coupled receptors known to be involved in MSI/MSE have been shown to be present and functional in this preparation, it seems reasonable to ask whether other postsynaptic Gq-type receptors might also be present and capable of mediating retrograde inhibition. In other words, is Gq-coupled retrograde inhibition a more general phenomenon, or is it limited to mGluR (Group I) and muscarinic receptors? Several Gq-coupled receptors have been described in the hippocampus, among them substance P, cholecystokinin (CCK), 5-HT2, bradykinin, lysophosphatidic acid, orexin A, histamine and 1 adrenergic (1-AR) receptors. Accordingly, we tested ligands for each of these receptors (see Table 1).
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In addition, we tested the same agonists in identified Type O neurons. Here the results were essentially identical (Table 1). It should be noted that PE did not inhibit EPSCs at 1 µM, but did so readily at 50 µM (EPSC inhibition by PE, 0.68 ± 0.04, n
= 19) in Type O (but not regular) neurons. This inhibition is likely to be presynaptic, as evidenced by an increase in the paired-pulse ratio (1st pair, 0.75 ± 0.07; 2nd pair, 0.93 ± 0.05, n
= 6; P < 0.005 by paired t test). However, we do not believe that this is due to action at the 1-AR because the action of PE was not blocked by prazosin at concentrations selective for the 1-AR (< 3 µM, data not shown). Additionally, treatment with pertussis toxin (300–500 ng ml–1), which ADP-ribosylates Gi/o-type subunits, thereby selectively blocking Gi/o signalling, blocked the PE response (PE inhibition (50 µM), 0.94 ± 0.02 n
= 6, P < 0.0001 versus PE control, unpaired t test), which is also inconsistent with direct action via a Gq-coupled receptor. The effect was also CB1-independent, persisting in CB1–/– cultures and in the presence of SR141716 (data not shown). The action does not appear to occur via the 2-AR because co-application of the 2-AR antagonist yohimbine failed to block the effect of PE even at 10 µM (same cell experiments: PE (15–50 µM), 0.75 ± 0.03; PE + yohimbine (1–10 µM), 0.76 ± 0.04, n
= 6, P > 0.05 paired t test). The PE may be acting via 
-AR or some other unknown receptor.
Lastly, we tested abnormal cannabidiol (abn-cbd) a compound that is structurally related to classical cannabinoids and that has been reported to act via an unknown Gq-coupled receptor (Offertaler et al. 2003). Rather than inhibiting EPSCs, abn-cbd modestly potentiated them (1 µM abn-cbd, 1.05 ± 0.02, n = 5; 10 µM abn-cannabidiol, 1.12 ± 0.04, n = 3). Although a potentiation is inconsistent with a role mediating MSE, it raises the possibility that abn-cbd does have a site of action in these neurons – perhaps even an endogenous receptor – and that abn-cbd and similar compounds may enhance neurotransmission.
MSE is distinct from DSE
The presence of both DSE and MSE in the same simple preparation allows us to explore MSE–DSE interactions. In principle, DSE might simply be the expression of depolarization-induced release of acetylcholine or glutamate acting at muscarinic or mGluRs, respectively. Also, it has been reported that hippocampal DSI is diminished by non-specific blockers of mGluRs (Varma et al. 2001; but see Kim et al. 2002). Consequently, we tested whether antagonists for either receptor group blocked DSE. Treatment with the non-selective muscarinic antagonist atropine (1 µM, 10 min) had no effect on DSE, indicating that muscarinic receptor activation is not necessary for DSE (DSE, 0.48 ± 0.07; DSE + atropine, 0.40 ± 0.06, n = 4). Similarly, 4-carboxyphenylglycine (4-CPG), a mGluR (Group I, 50 µM) antagonist, did not inhibit DSE in same-cell experiments where DSE was elicited first, and again in the presence of 4-CPG (DSE control, 0.65 ± 0.07; DSE/4-CPG, 0.68 ± 0.08, n = 5). Furthermore, neither antagonist affected EPSC amplitude (data not shown), indicating that there is no tonic mGluR- or muscarinic receptor-dependent endocannabinoid release by these neurons.
MSE occludes DSE
MSE sets in motion cellular machinery that produces an endocannabinoid that acts at presynaptic CB1 receptors. We have previously shown that in these neurons DSE is most probably mediated by 2-AG (Straiker & Mackie, 2005). If MSE occurs using the same endocannabinoid, one would expect occlusion of MSE by 2-AG and occlusion of DSE by MSE. We found that in cells treated separately with oxo-M (5 µM) and 2-AG (5–10 µM), co-application of the two did not result in inhibition that exceeded that induced by 2-AG alone (Fig. 5A; same-cell experiments: oxo-M, 0.76 ± 0.04; 2-AG, 0.56 ± 0.09; 2-AG plus oxo-M, 0.63 ± 0.09, n = 5). These results suggest that 2-AG occludes MSE.
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Do MSE and DSE synergize?
Several studies have now described a ‘DSI-enhancement’ whereby simultaneous subthreshold DSI- and MSI-inducing stimuli result in synergistic IPSC inhibition (Varma et al. 2001). The proposed implication is that endocannabinoids act as coincidence detectors (Hashimotodani et al. 2005) integrating membrane depolarization and PLC 1 activation. Does MSE similarly enhance DSE in a correlate of ‘DSI enhancement’? To explore this question, we performed a similar set of experiments in which 50 nM oxo-M – which does not inhibit EPSCs – was coupled with a DSE stimulus that resulted in less than a 20% inhibition (determined by testing 0.05, 0.1, 0.2, 0.3, 0.5 and 1.0 s depolarizations until a 20% inhibition of EPSCs was reached – the preceeding shorter stimulus duration was then chosen). We found that the duration of depolarization required to induce threshold DSE was generally a few hundred milliseconds (median, 300 ms); however, 10% of neurons exhibited substantial DSE with only 50 ms depolarizations (Fig. 6A). We found that subthreshold MSE coupled with modest DSE consistently yielded synergistic inhibition (Fig. 6B; same-cell experiments: threshold DSE, 0.88 ± 0.02; oxo-M (50 nM), 0.97 ± 0.03; DSE + oxo-M, 0.78 ± 0.04; n
= 10, P < 0.05, paired t test), an apparent ‘DSE enhancement’. As a control, we paired DSE stimuli at the same threshold level but in the absence of drug. This did not result in an enhancement (data not shown).
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It is interesting that whereas DHPG mediates MSE in type O neurons, we did not observe comparable DSE potentiation in these neurons (Fig. 6D; same cell experiments: threshold DSE, 0.92 ± 0.01; DHPG (2 µM), 0.96 ± 0.05; DSE + DHPG, 0.87 ± 0.02, n = 4, P > 0.05, paired t test DSE versus DSE/DHPG). This indicates that DSE enhancement is not necessarily present even if the machinery for both DSE and MSE are present in the same autaptic neuron.
A time-dependent DSE potentiation
In the course of our experiments, we noted that DSE often appeared to exhibit potentiation: if DSE was elicited twice, the degree of the second DSE was typically greater. It is interesting that the subsequent DSE remained stronger, suggesting that sensitization might be taking place. On further investigation we identified that the determining factor in this potentiation is time. Potentiation took place over the course of 3 min after entering the whole-cell configuration of patch-clamp recording, and is thus presumably a result of neuronal dialysis (i.e. it is a recording artifact) (Fig. 7; DSE at 0–1 min, 0.62 ± 0.08, n
= 9; 1–3 min, 0.46 ± 0.03, n
= 7; 3–6 min, 0.32 ± 0.03 n
= 16; 6+ min, 0.37 ± 0.04, n
= 11).
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Interactions among MSE receptors
Receptors mediating MSE have been studied independently (Maejima et al. 2001; Kim et al. 2002), but do these receptors interact? Do they occupy discrete cellular compartments, perhaps incorporating distinct signalling pathways? Do they act cooperatively, antagonistically or synergistically?
Application of saturating concentrations of oxo-M generally produces inhibition comparable to (though somewhat less than) DSE in the same cell. Will activating mGluR and muscarinic receptors simultaneously increase inhibition? We found that co-application of saturating concentrations of oxo-M (5–10 µM) with DHPG (50–100 µM) produced only a modestly (and statistically non-significant) increased MSE when compared to their individual application (Fig. 8A; oxo-M, 0.65 ± 0.06; DHPG, 0.77 ± 0.05; oxo-M + DHPG, 0.63 ± 0.06, n = 8). This result implies that the apparent ceiling on MSE is not due to a compartmentalization of postsynaptic Gq-coupled receptors, which could then independently release endocannabinoids.
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Lastly, we have shown above that extended application of oxo-M induces desensitization of MSE. Is this oxo-M desensitization homologous (i.e. limited to muscarinic MSE), or does it also extend to mGluR (group I)-mediated MSE? We found that oxo-M in fact also cross-desensitizes DHPG-induced MSE in regular neurons (Fig. 8C; DHPG inhibition of EPSCs in regular neurons desensitized by oxo-M, 0.99 ± 0.06, n = 6). As a control, we performed the same experiment in Type O neurons. As oxo-M-induced inhibition in Type O neurons does not represent a form of MSE, the inhibition should not (and did not) induce postsynaptic desensitization of the MSE response (Fig. 8C, DHPG inhibition of EPSCs in oxo-M-treated Type O neurons, 0.74 ± 0.02, n = 4). That MSE desensitization is heterologous has considerable implications when considering the interactions between glutamatergic and muscarinic signalling. Several mechanisms could conceivably bring about such a desensitization, including receptor [mGluR metabotropic acetylcholine receptor (mAChR)] cross-desensitization or desensitization at either the PLC or the DAG lipase level.
9-THC antagonizes MSE
9-THC has been shown to attenuate DSE in autaptic hippocampal neurons in a dose-dependent fashion, probably by antagonizing 2-AG at CB1 receptors. Does 9-THC also attenuate MSE? To test this we elicited MSE before and during treatment with 9-THC. Notably, we found that at 200 nM, 500 nM and 5 µM
9-THC progressively antagonized MSE induced by 2 µM oxo-M. In contrast, 50 nM
9-THC did not noticeably interfere with MSE (Fig. 8D; control MSE, 0.56 ± 0.06; 9-THC (50 nM)/MSE, 0.44 ± 0.12, n
= 3; 9-THC (200 nM)/MSE, 0.80 ± 0.08, n
= 3; 9-THC (500 nM)/MSE, 0.91 ± 0.07, n
= 4; 9-THC (5 µM)/MSE, 1.04 ± 0.07, n
= 3; P > 0.05 versus 50 nM THC, P < 0.1 versus 200 nM THC, P < 0.05 versus 500 nM THC, P < 0.001 versus 5 µM THC, one-way ANOVA with Dunnett's post hoc test). Consistent with previous results (Straiker & Mackie, 2005), 9-THC did not inhibit EPSCs when applied alone.
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Discussion |
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9-THC. With the presence of both DSE and MSE, we have found that the autaptic neuron serves as a powerful model system to dissect endogenous cannabinoid signalling. Shared signalling machinery of MSE and DSE
One important question regarding MSE is what components, if any, this retrograde signalling pathway shares with DSE. In principle, MSE could occur via separate endocannabinoid production, or even a separate subpopulation of presynaptic cannabinoid receptors. However, given what is known of MSE and DSE, sharing of components seems likely. DSE and MSE in these neurons share at least a partial requirement for filled internal calcium stores. Occlusion and knockout experiments tell us that MSE and DSE employ the same presynaptic receptors. Of the serious candidates as mediators of DSE and MSE (2-AG, anandamide and noladin ether) our experiments suggest that only 2-AG could be the actual mediator of MSE in these neurons. Anandamide and noladin ether both fail to reversibly inhibit EPSCs (Straiker & Mackie, 2005) and by this criterion are unlikely to be the candidate endocannabinoid. 2-AG, on the other hand, closely mimics both DSE and MSE. The only alternative is invoking a novel endocannabinoid, a possibility that cannot be excluded by our experiments. If 2-AG does in fact mediate both MSE and DSE, and considering the concentration–response curve for inhibition by 2-AG of EPSCs, then the maximal activation of MSE brings about an effective local synaptic 2-AG concentration approaching 5 µM (Straiker & Mackie, 2005).
Where MSE and DSE part ways
The above similarities between MSE and DSE are nor surprising; of greater interest are the differences between the two. Most notably, MSE desensitizes over the course of minutes whereas DSE does not. As such, and because presynaptic receptor sensitivity does not change, the site of desensitization must be postsynaptic, and not directly involve CB1 receptors. The heterologous desensitization observed makes muscarinic receptor or mGluR tachyphylaxis unlikely. Instead, it appears most likely that the desensitization occurs after mGluR or muscarinic receptor activation, but early in the Gq signalling pathway, perhaps by desensitization of PLC- or DAG lipase or via depletion of precursors.
DSE is a widespread mechanism for feedback inhibition and several forms of its regulation are becoming apparent. For example, CB1 receptor desensitization makes possible downregulation of presynaptic sensitivity to DSE. Heterologous MSE desensitization adds two additional sites (muscarinic and metabotropic glutamate signalling) at which adaptation might occur. It remains to be shown that MSE desensitization occurs outside of the autaptic hippocampal preparation, although it should be noted that treatments of comparable duration inducing muscarinic MSI in hippocampal slices did not result in apparent desensitization (Edwards et al. 2006). If MSE desensitization is shown to occur in vivo, its mechanism might also serve as a pharmacological target to modulate endocannabinoid signalling.
The observed inhibition of MSE signalling by the Gq-PLC uncoupler U73122 is consistent with MSE action via PLC, but these results diverge from those seen recently for DSI in the hippocampus (Edwards et al. 2006). The difference between our results and those of Edwards and coworkers – who saw no inhibition of DHPG-mediated MSI by U73122 – may be explained by differences in experimental preparation or may point to an underlying difference in endocannabinoid release mechanisms for MSE versus MSI.
Threshold MSE and DSE act synergistically
The implications of interactions between MSE and DSE significantly expand the importance of endocannabinoids as neuromodulators. In inhibitory conventional hippocampal cultures when examining MSI and DSI, the co-application of two subthreshold stimuli induced a synergistic inhibition of IPSCs (Ohno-Shosaku et al. 2002a; Hashimotodani et al. 2005). The comparable DSE–MSE synergism described here expands this phenomenon to glutamatergic synapses and to mGluRs. This might be a mechanism for integration of fast (ionotropic) glutamate signalling and slow muscarinic signalling in hippocampal function.
A neuronal subpopulation with a differential MSE complement
The idea of neuronal subpopulations in autaptic cultures is not novel – subpopulation effects have been described before (e.g. Straiker et al. 2002). However, the reliable identification of such a subpopulation, in this case the ‘Type O’neurons, has implications for future studies making use of autaptic or conventional hippocampal neurons. We make no claim regarding the nature of these populations. The neurons are cultured from CA1–CA3 of hippocampi from postnatal animals in which considerable differentiation has already occurred. As such they might represent developmentally delayed forms of neurons found in vivo, but the subpopulations could also arise as culturing artifacts. The current studies of CB1-dependent signalling do not appear to be affected by this heterogeneity. However, the presence of differential MSE across neuronal subpopulations will enable contrasting studies of MSE. The enhanced MSE induced by the same concentrations of DHPG (without a corresponding difference in DSE) in Type O neurons suggests that the (group I) mGluRs in specific neuronal populations are differentially coupled to endocannabinoid-producing machinery, at least in part.
9-THC antagonizes MSE
Previously we have shown that 9-THC antagonizes endocannabinoid signalling in autaptic hippocampal neurons by two very different means. 9-THC not only acutely antagonizes DSE but also downregulates the CB1 receptor with chronic exposure. Our finding that 9-THC also acutely antagonized MSE is consistent with these findings and again raises the question of whether a major mechanism for acute 9-THC action in the brain may be by antagonism of 2-AG signalling.
Summary
Metabotropic receptor-stimulated release of endocannabinoids is well established; the focus of research is now on the working details and the functional implications of this process (e.g. MSI/MSE). Now we have described a hippocampal MSE, which is qualitatively distinct from MSI. With a neuronal preparation expressing both MSE and DSE within a single neuron, we have gained insight into the interactions between MSE and DSE as well as points of desensitization. Identification of such means of potential adaptation is a crucial next step in characterizing the precise workings of endogenous cannabinoid signalling in excitatory neurons. CB1 receptor desensitization represents a presynaptic means of modulating endocannabinoid-mediated retrograde inhibition. The family of MSE-related effects reported here, including MSE desensitization and cross desensitization, serve as additional postsynaptic mechanisms whereby multiple, disparate inputs can separately or in combination modulate endocannabinoid-mediated plasticity. Certainly, the combination of MSE and DSE offers a powerful, flexible means for postsynaptic metabotropic receptor modulation of upstream ionotropic glutamatergic signalling. Finally, the antagonism of the endogenous cannabinoid signalling system by the exogenous agonist 9-THC appears to be a common theme for disparate forms of endocannabinoid-mediated plasticity.
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References |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Edwards DA, Kim J & Alger BE (2006). Multiple mechanisms of endocannabinoid response initiation in hippocampus. J Neurophysiol 95, 67–75.
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), CB1–/– neurons (
) and in presence of CB1 antagonist SR141716 (100 nM; 
). C, top panel shows comparison of paired-pulse ratios (interval, 60 ms) before and after MSE stimulus. Bottom panel shows representative traces (scale bars, 1 nA and 10 ms). Traces are normalized to the 1st EPSC of the control stimulus to facilitate comparison. D, comparison of oxo-M responses (5–10 µM) and saturating DSE responses in the same cell (n
= 14). E, left panel shows comparison of magnitude of response to serial oxo-M treatments in the same cell (n
= 4). Right panel shows sample timecourse with brief, sequential oxo-M treatments. F, graph showing inhibition of MSE (oxo-M) after pretreatment with Ca2+ ATPase inhibitor thapsigargin (1 µM, 20+ min; n
= 14). G, comparison of MSE under control conditions and after treatment with PLC inhibitor U73122 (3 µM, 2.5–5 min).
) and in presence of oxo-M (5 µM, 
). C, left panel shows a summary of oxo-M/DSE occlusion experiments (same cell) with inhibition by DSE alone and partial occlusion of DSE by oxo-M-induced MSE (n
= 6). Right panel shows comparable partial occlusion of DSE by DHPG-mediated MSE in Type O neurons (n
= 4). **P < 0.005, paired t test.
