In this study, Zhou et al. address the pertinent question of whether long-term potentiation (LTP) induced by withdrawal from opioids at synapses of primary afferent nerve fibers might be induced presynaptically (Zhou et al., 2010). LTP after opioid withdrawal was originally described in our Science report (Drdla et al., 2009). In that study, we identified a novel form of LTP at synapses between primary afferent C-fibers and n...
In this study, Zhou et al. address the pertinent question of whether long-term potentiation (LTP) induced by withdrawal from opioids at synapses of primary afferent nerve fibers might be induced presynaptically (Zhou et al., 2010). LTP after opioid withdrawal was originally described in our Science report (Drdla et al., 2009). In that study, we identified a novel form of LTP at synapses between primary afferent C-fibers and neurons in lamina I of the spinal cord dorsal horn. This form of LTP was induced by abrupt withdrawal from µ-opioid receptor (MOR) agonists. We concluded that the induction of LTP by opioid withdrawal was postsynaptic because it required activation of postsynaptic G-protein-coupled receptors, postsynaptic activation of NMDA receptors, and rise in postsynaptic calcium concentration.
In their paper, Zhou et al. make essentially two predications: First, they provide additional data on opioid withdrawal LTP and conclude that withdrawal LTP is a presynaptic event resulting from increased presynaptic glutamate release from TRPV1-expressing primary afferents. Second, they suggest that there were several problems in our study design.
In the following, we address these points.
I. Does LTP originate presynaptically from C-fiber afferent terminals?
Zhou and colleagues recorded EPSCs from lamina I and II neurons in response to electrical stimulation of the dorsal root. Inspection of the original traces (in Figs. 1-4, 6 and 7), however, indicate that the latencies between the stimulation artefact and the synaptic responses are extremely short in all cases and cannot be quantified adequately. This suggests that electrical stimulation was done very close to the recording site (probably at or near the dorsal root entry zone). In any case, with the experimental set-up used by Zhou et al. in this and in previous studies, it is virtually impossible to securely measure monosynaptic C-fiber-evoked responses as neither the response latency nor the jitter can be assessed with sufficient precision. This was acknowledged by the same authors in a previous publication (Li et al., 2002) and it is also obvious from their present work.
In search of potential presynaptic mechanisms, Zhou et al. chose various approaches: 1.) They recorded miniature EPSCs (mEPSCs) and found that the rate of mEPSCs was depressed during DAMGO application and potentiated after withdrawal. Synapses from primary afferent nerve fibers constitute only a small fraction of the total excitatory input to spinal dorsal horn neurons. mEPSCs monitor the activity of unknown types of excitatory synapses impinging on the neuron under study and thus cannot be used to assess presynaptic expression of LTP at the small subpopulation of synapses derived from primary afferent C-fibers. 2.) They used a paired-pulse protocol and state that “LTP was associated with an increased paired-pulse ratio (Fig. 1A and B)”. An increase in paired-pulse ratio is, however, typically not associated with presynaptic expression of LTP, because it indicates that synaptic release probability is reduced, not enhanced (see for example, de Armentia and Sah, 2007). From the data provided by Zhou et al., no firm conclusions can be drawn as to the pre- or postsynaptic expression of opioid withdrawal LTP. This is unfortunate, because it would have been of considerable interest to show that the postsynaptic induction of opioid withdrawal LTP demonstrated in our report (Drdla et al., 2009) is complemented by a presynaptic expression.
II. Are the data in our Science report insufficient to demonstrate that induction of LTP upon opioid withdrawal involves postsynaptic mechanisms?
Zhou et al. make three statements here:
1.) They suggest that the power in some of our experimental groups was too low and results would be prone to sampling errors. Our data showed that in 10 of 14 neurons, withdrawal from opioids induced LTP. If induction of withdrawal LTP were entirely presynaptic as suggested by Zhou et al., then in our report neither postsynaptic GDP-beta-S, postsynaptic BAPTA, caged BAPTA, nor postsynaptic MK-801 should have blocked LTP induction upon opioid withdrawal in any of the neurons tested in our study. It can be calculated that the combined probability of sampling errors in all our experimental groups is p <0.0001.
2.) They argue that our recording condition (holding potential at –70 mV, 2 mM MgCl2 in the pipette solution, and 1.3 mM MgCl2 in the bath solution was suboptimal for studying postsynaptic NMDA receptors. From the literature (e.g., South et al., 2003) and from our own unpublished data, however, it is obvious that in spinal dorsal horn neurons NMDA receptor-mediated currents can be elicited under the near physiological conditions that we used in our report (Drdla et al., 2009). Opioids may activate PKC, and NMDA receptor-mediated currents are further potentiated by activators of PKC. This led us to the hypothesis discussed in our report: “NMDA receptors have a PKC phosphorylation site and phosphorylation removes the voltage-sensitive Mg2+ block. This may lead to NMDA receptor activation and Ca2+ influx at or near the resting membrane potential in the absence of any presynaptic activity, possibly by glial cell-derived glutamate or elevated glutamate levels during opioid withdrawal.” Furthermore, it is well established that the voltage- dependence of the Mg2+ block depends upon the subunit composition of NMDA receptors. Receptors composed of NR1/NR2D subunits are considerably less Mg2+-sensitive than NR1/NR2A or NR1/NR2B receptors (Qian et al., 2005). Importantly the poorly Mg2+-sensitive NR2D NMDA receptors are expressed in lamina I neurons (Tong et al., 2008).
3.) In our study, we used MK-801 at a concentration of 10 µM in the pipette solution to study whether activation of NMDA receptors in the postsynaptic neuron was required for the induction of opioid withdrawal LTP. In the literature, MK-801 is used at concentrations ranging from 5 to 1000 µM in the pipette solution (Garraway et al., 2003;Arvanov et al., 1999;Arvanov et al., 2000;Humeau et al., 2003). Unpublished data from our group unequivocally show that MK-801 applied at a concentration of 10 µM to the pipette solution significantly blocks NMDA receptor-mediated currents in lamina I neurons. Zhou et al. argue, however, that "1000 µM MK-801 in the pipette solution is required to block postsynaptic NMDA receptors through intracellular dialysis” citing work by Bender et al. (2006) and Humeau et al. (2003). This is, however, a misquotation of their work as these authors had not studied which concentrations of MK-801 are required for blocking NMDA receptors and they had not shown that MK-801 at 10 µM would be ineffective.
Acknowledgements
Supported by grant # P18129-B02 from the Austrian Science Fund (FWF).
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