Differential modulation of nociceptive versus non-nociceptive synapses by endocannabinoids
© Higgins et al.; licensee BioMed Central Ltd. 2013
Received: 14 December 2012
Accepted: 28 May 2013
Published: 1 June 2013
Although a number of clinical and preclinical studies have demonstrated analgesic effects of cannabinoid treatments, there are also instances when cannabinoids have had no effect or even exacerbated pain. The observed pro-nociceptive effects appear to be due to cannabinoid-induced disinhibition of afferent synaptic input to nociceptive circuits. To better understand how cannabinoid-mediated plasticity can have both pro- and anti-nociceptive effects, we examined the possibility that cannabinoids differentially modulate nociceptive vs. non-nociceptive synapses onto a shared postsynaptic target. These experiments were carried out in the central nervous system (CNS) of the medicinal leech, in which it is possible to intracellularly record from presynaptic nociceptive (N-cell) or pressure-sensitive (P-cell) neurons and their shared postsynaptic targets.
The endocannabinoid 2-arachidonoyl glycerol (2AG) elicited significant long-lasting depression in nociceptive (N-cell) synapses. However, non-nociceptive (P-cell) synapses were potentiated following 2AG treatment. 2AG-induced potentiation of non-nociceptive synapses was blocked by the TRPV antagonist SB366791, suggesting involvement of the same TRPV-like receptor that has already been shown to mediate endocannabinoid-dependent depression in nociceptive inputs. Treatment with the GABA receptor antagonist bicuculline also blocked 2AG-induced potentiation, consistent with the idea that increased synaptic signaling was the result of endocannabinoid-mediated disinhibition. Interestingly, while bicuculline by itself increased non-nociceptive synaptic transmission, nociceptive synapses were depressed by this GABA receptor antagonist indicating that nociceptive synapses were actually excited by GABAergic input. Consistent with these observations, GABA application depolarized the nociceptive afferent and hyperpolarized the non-nociceptive afferent.
These findings show that endocannabinoids can differentially modulate nociceptive vs. non-nociceptive synapses and that GABAergic regulation of these synapses plays an important role in determining whether endocannabinoids have a potentiating or depressing effect.
Endogenous cannabinoids (endocannabinoids or eCBs), such as anandamide and 2-arachidonoyl glycerol (2AG), are lipid neurotransmitters found throughout the central nervous system of both vertebrates and invertebrates that modulate a number of behavioral processes including appetite, cognition, emotion, sensory processing and nociception [1, 2]. In many regions of the CNS, the primary physiological effect of eCBs is depression of synaptic transmission that can be short-term (milliseconds to seconds) or long-term (tens of minutes to hours) . eCBs can depress both excitatory and inhibitory synapses, so that from a functional standpoint these neurotransmitters can be bi-directional modulators of neural circuits depending on whether there is depression of an excitatory pathway (decreasing circuit activity) or depression of an inhibitory pathway (increasing circuit activity via disinhibition). This is a significant consideration in terms of development of eCB-based pharmacotherapies because treatments focused on depression of excitatory synapses may produce undesired effects through depression of inhibitory synapses. The importance of this issue has become apparent in the potential use of cannabinoid-based treatments for chronic pain. In a recent study, cannabinoids were found to reduce GABAergic and glycinergic inhibitory signaling in the spinal cord that contributed to secondary hyperalgesia due to disinhibition of afferent synaptic input to spinal pain circuits . However, it was not known whether this pro-algesic effect occurred at the nociceptive afferent synapses (Aδ and C fibers) or at non-nociceptive afferent synapses (Aβ fibers). Non-nociceptive afferents can have input to pain circuits in the spinal cord as a result of central sensitization and can potentially contribute to chronic pain conditions .
The leech CNS is known to contain the eCBs 2AG and anandamide with 2AG being the more abundant transmitter . Furthermore, 2AG has been shown to mediate long-term depression (LTD) in leech nociceptive synapses that are remarkably similar to mammalian eCB-LTD in terms of cellular mechanisms [3, 10]. Like other protostomal invertebrates, the leech lacks orthologues to the vertebrate cannabinoid receptors CB1 and CB2 . However, transient potential receptor vanilloid (TRPV) channels can also act as cannabinoid receptors [15–18] and previous pharmacological studies from our laboratory indicate that a leech TRPV-like receptor mediates 2AG-induced depression in nociceptive synapses .
In the present study, the effects of 2AG on nociceptive synapses (those made by the N-cells) versus non-nociceptive synapses (those made by the P-cells) were compared in which both afferent types converged on the same postsynaptic target (the longitudinal or “L” motor neuron). 2AG elicited depression at nociceptive synapses, but potentiation at non-nociceptive synapses. This 2AG-mediated potentiation of the non-nociceptive synapses required functional GABA receptors, indicating eCB-induced increases in synaptic signaling were the result of synaptic disinhibition.
Effects of 2AG on nociceptive vs. non-nociceptive synapses
To determine if these opposing effects of 2AG were a feature of other nociceptive and non-nociceptive synapses, N- and P-cell input to the anterior pagoda (AP) cell was also examined. Although the function of the AP cell is not known, the monosynaptic, glutamatergic P-to-AP synapses have been used to study a variety of forms of synaptic plasticity in the leech [21–25]. However, the N-to-AP synapse had not been previously characterized and this was done prior to conducting the 2AG experiments. In general the N-to-AP synapse was quite similar to the N-to-L connection. N-to-AP was substantially reduced by CNQX (20 μM) indicating glutamatergic transmission (Additional file 1: Figure S1A). In experiments with high MgCl2 saline (15 mM), which blocked all chemical synaptic transmission , the majority of the N-to-AP EPSP was eliminated except for a small (<1 mV) early component that appeared to be a rectifying electrical synapse between the N-and AP-cell (Additional file 1: Figure S1B). This putative electrical EPSP was also observed in the CNQX experiments. Experiments with high divalent saline (18 mM MgCl2/15 mM CaCl2) eliminated later components of the N-to-AP EPSP indicating the presence of polysynaptic elements (Additional file 1: Figure S1C). While positive current associated with the N-cell action potential did propagate to the AP via electrical coupling, negative current injected into the N-cell did not spread to the AP (data not shown). Interestingly, evidence of electrical coupling was also observed in the AP-to-N direction. Injection of 500 msec current pulses showed that negative current, but not positive current, could spread from the AP to the N-cell (Additional file 1: Figure S1D).
In terms of eCB modulation, 2AG (100 μM) depressed N-to-AP synapses (n=9 and 5 respectively; t=2.91, p≤0.05), but potentiated P-to-AP synapses relative to controls (n=6 and 5 respectively; t=2.36, p≤0.05) identical to N- and P-cell inputs to the L motor neuron (Figure 2C). No obvious changes in postsynaptic IR were observed during the P-to-AP (2AG group=111.5±3.2%, control=106.8±5.6) or N-to-AP (2AG group=104.6±3.8%, control=97.7±6.2%) synapses.
Role of GABA receptors during 2AG-induced potentiation
eCBs are known to depress inhibitory synaptic transmission and eCB-induced disinhibition has been shown to increase synaptic signaling within spinal nociceptive circuits, although the identity of the presynaptic input(s) being enhanced was not known . Therefore, we examined the potential role of GABA-A receptors during 2AG-induced potentiation of the non-nociceptive synapse in the leech. The leech CNS contains GABAergic neurons and the GABA-A receptor antagonist bicuculline has been shown to disinhibit other circuits that utilize P-cell input [26–28]. To test whether functional GABA-A receptors are required for 2AG-mediated potentiation, ganglia were pre-treated with bicuculline (100 μM) followed by the pre-test measurements of the P-to-L EPSP, 15 min application of 2AG, washout for 60 mins and finally post-test EPSP measurements. The 2AG treatment that normally produces potentiation was blocked in the non-nociceptive P-to-L synapses that had undergone bicuculline+2AG treatment (Figure 3B; n=5). P-to-L synapses that were pre-treated with bicuculline, but subsequent 2AG treatment omitted, were unchanged between the pre- and post-tests (Figure 3B, n=5). One-way ANOVA of the 2AG+bicuculline, bicuculline, 2AG and vehicle control group showed a significant effect of treatment (F2,24=7.13, p≤0.005). Post-hoc analysis showed that the 2AG group was statistically different from the control group (p<0.01) and that the 2AG+bicuculline and bicuculline groups were not. No change in postsynaptic input resistance was observed in the 2AG+bicuculline (104±4.4%) or bicuculline only group (96±4.6%). These findings support the hypothesis that 2AG-induced potentiation of non-nociceptive synapses is mediated by disinhibition as a result of a decrease in GABAergic input.
Next, P-to-L and N-to-L EPSPs were recorded prior to and then during acute bicuculline treatment (100 μM for 10–15 mins). As expected, the P-to-L EPSP amplitude increased during bicuculline treatment (n=7) compared to control synapses (n=5); that is, disinhibition of the non-nociceptive synapse was observed (Figure 4B; t=2.71, p≤0.05). However, bicuculline treatment actually decreased the amplitude of the N-to-L EPSP (n=8) compared to control synapses (n=5) suggesting that GABA has a tonic excitatory effect on this nociceptive synapse (Figure 4B; t=2.35, p≤0.05). No change in postsynaptic input resistance was observed during either the P-to-L synapse (bicuculline=96.7±4.9, control=105.7±6.4%) or N-to-L synapse (bicuculline=107.7±2.2%, control=98.7±3%) experiments. These findings are consistent with the idea that this nociceptive synapse does not undergo 2AG-induced disinhibition, at least in part, because the presynaptic neuron is excited, not inhibited, by GABA.
The endocannabinoid 2AG was found to differentially modulate nociceptive vs. non-nociceptive synapses in the leech, depressing the former and potentiating the latter. 2AG-induced potentiation of non-nociceptive synapses was blocked by the TRPV1 inhibitor SB366791, suggesting that the involvement of a TRPV-like receptor. In mammals, the TRPV1 receptor has been shown to be an important cannabinoid receptor that mediates synaptic depression in a variety of brain regions [16–18, 30]. Protostomal invertebrates lack orthologues to the vertebrate CB1/CB2 receptors  and we have previously proposed that TRP channels, such as TRPV, may represent the earliest endocannabinoid receptors in the animal kingdom [10, 11]. One critical difference between the nociceptive and non-nociceptive synapses is that while N-cells appear to possess TRPV-like receptors, P-cells do not . This would indicate that the effects of 2AG on P-cell synapses are indirect and mediated by an unknown, 2AG-sensitive neuron. It is unlikely that the postsynaptic L motor neuron is being directly modulated by 2AG because it too lacks TRPV-like receptors, although it does appear to be capable of synthesizing 2AG .
The fact that 2AG did not potentiate nociceptive synapses appears to be due to differences in how GABA affects nociceptive vs. non-nociceptive synapses (Figure 5). The same bicuculline treatment that disinhibited non-nociceptive synapses actually decreased N-cell synaptic transmission suggesting that GABA has an excitatory effect on these nociceptive synapses. This is supported by the observation that GABA applied to the soma depolarizes the lateral N-cell . In the present study, the ability of GABA to depolarize the N-cell was replicated and extended by showing that this GABA-induced depolarization could be inhibited by bicuculline. Furthermore, direct application of GABA to the P-cell was found to elicit hyperpolarization that was also inhibited by bicuculline. The excitatory effects of GABA on the N-cell are likely the result of elevated intracellular Cl- concentration so that the Cl- equilibrium potential is depolarized relative to the resting potential. Thus, activation of ionotropic GABA receptors would cause a Cl- efflux, depolarizing the N-cell. It well-established that GABA can depolarize nociceptive afferents in the spinal cord as a result of elevated intracellular Cl- concentrations/Cl- equilibrium potential (ECl) . GABA-mediated depolarization has been shown to increase synaptic transmission in both the brain and the spinal cord circuits [32–35], although there are also instances when GABA-mediated depolarization inhibits synaptic transmission via current shunts or Na+ channel inactivation [31, 35].
It is not clear whether GABAergic modulation of the P-to-L synapse is being exerted on the presynaptic or postsynaptic neuron (or both). Leech motor neurons do receive inhibitory GABAergic input [26, 36], but no changes in postsynaptic input resistance were observed during any of the 2AG experiments, which would argue against a postsynaptic mechanism. Furthermore, the fact that N- and P-cell synapses onto the same postsynaptic target (L motor neuron or AP cell), but underwent opposite changes following 2AG treatment would also support a presynaptic mechanism for both eCB-induced potentiation and depression. Previous studies have been supportive of a presynaptic mechanism mediating 2AG-induced depression of nociceptive (N-to-L) synapse [10, 11]. Nevertheless, the possibility of localized, synapse-specific changes in the postsynaptic neuron cannot be eliminated and additional studies are required to further assess the role of pre- vs. postsynaptic modulation during eCB-mediated synaptic plasticity.
The results from this study address important issues regarding the modulatory effects of eCB at the microcircuit level. It is well-established that eCBs depress inhibitory (as well as excitatory) synaptic transmission and that such eCB-mediated disinhibition is likely to have important consequences in terms of neural circuit function [16, 37–42]. For example, eCB-induced disinhibition has been observed to lower the threshold for initiating LTP [43–45]. However, this is the first time, to our knowledge, that eCB-induced increases in evoked synaptic transmission via disinhibition have been directly observed. Furthermore, by using the well-described CNS of the leech it was possible to observe opposing synaptic effect of eCBs (depression vs. potentiation) on distinct afferent inputs that converge onto a shared postsynaptic target. This allowed us to address the issue as to why eCB-induced disinhibition does not lead to increased synaptic signaling in more neurons given the widespread nature of GABAergic regulation of synaptic transmission. We found evidence that while some synapses were sensitive to eCB-mediated disinhibition; others were “protected” from such modulation because they were not inhibited by GABA. In fact, GABA appeared to have an excitatory effect on these synapses based on results from experiments in this study and those from Sargeant et al. , presumably as a result of elevated intracellular Cl- levels. This would suggest that the Cl- gradient in neurons plays a critical in regulating the effects of eCBs on synaptic signaling. Such a mechanism has already been suggested by Christie and Mallet .
These findings may contribute to understanding why there is such conflicting evidence from both animal model and clinical studies regarding the efficacy of cannabinoid based analgesic therapies [46–50]. In some clinical studies, cannabinoids were found to be ineffective and could even enhanced pain associated with evoked mechanical stimuli resulting in mechanical hyperalgesia or allodynia [4, 46, 51]. One explanation for these pro-nociceptive effects is that eCBs have been shown to depress inhibitory synapses in the spinal cord, thereby disinhibiting spinal pain circuits, which contributes to the development of secondary mechanical hyperalgesia . It is possible that eCBs selectively enhance synaptic input from afferents that contribute to mechanical hyperalgesia or allodynia, such as Aβ fibers , but depress synaptic input from nociceptive afferents, such as Aδ or C fibers . As stated above, these synaptic potentiation vs. depression effects of eCBs may be due to whether GABA inhibits or excites the relevant afferent . Although the present study is focused on changes in afferent signaling, inhibitory and excitatory interneurons in the dorsal horn can also undergo shifts in the ECl that lead to GABA-elicited depolarization during neuropathic pain [52, 53]. Changes in GABAergic signaling onto these neurons may also impact how cannabinoid-based treatments affect these nociceptive neural circuits.
Nociception is a fundamental sensory process that exhibits considerable evolutionary conservation between vertebrates and invertebrates [54, 55]. The leech, in particular, provides a useful model system in which to study the basic physiological processes related to nociception. It possesses afferents innervating the skin that demonstrate clear nociceptive properties and share many features with vertebrate polymodal nociceptors including a high threshold for both mechanical and thermal (>40°C) stimuli as well as sensitivity to noxious chemical stimuli . These nociceptors are clearly distinguishable from the lower threshold non-nociceptive mechanosensory neurons and it is possible to carry out detailed recordings from nociceptive vs. non-nociceptive synapses.
The findings from the present study suggest that the effectiveness of cannabinoid-based analgesic therapies is likely to depend on the type of nociception that is being experienced. As already stated, there is evidence from both preclinical and clinical studies that eCBs can have both pro- and anti-nociceptive effects [4, 46]. Interestingly, there is also evidence to suggest that central TRPV receptor activation can have opposing effects on spinal nociceptive circuits in rodents mediating both synaptic disinhibition that resulted in allodynia  and persistent depression of C fiber evoked EPSPs [57–59]. Cannabinoid-based therapies may be appropriate for conditions that result from spontaneous activity from nociceptive afferents, such neuropathy-associated chronic pain [49, 60]. On the other hand, conditions that are dominated by mechanical hyperalgesia and/or allodynia may be insensitive to or even exacerbated by cannabinoid-based treatments due to the potential involvement of non-nociceptive afferents . Additional studies using the leech CNS, at both the synaptic and behavioral level, may contribute to better understanding the pro- vs. anti-nociceptive effects of eCBs.
Leeches (Hirudo verbana) were obtained from commercial suppliers (Leeches USA, Westbury, NY and Niagara Leeches, Cheyenne, WY) and maintained in artificial pond water (0.50g/L H2O Hirudo salt) on a 12 hour light/dark cycle at 18°C. Ganglia were dissected and pinned in a recording chamber with constant perfusion of normal leech saline (≈1.5 ml/min). All dissections and recordings were carried out in normal leech saline (110 mM NaCl, 4 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2, 5 mM NaOH, and 10 mM HEPES, pH=7.4). Drugs were dissolved in leech saline from stock solutions and final concentrations were made just prior to respective experiments. The following drug was obtained from Tocris (Ellisville, MO): 2-arachidonoyl glycerol (2AG). Drugs obtained from Sigma-Aldrich (St. Louis, MO) included CNQX, dimethyl sulfoxide (DMSO), and bicuculline.
Techniques used in this study have been described in detail in . Briefly, current clamp (bridge balanced) intracellular recordings were carried out using sharp glass microelectrodes (tip resistance 35–40 MΩ) made from borosilicate capillary tubing (1.0 mm OD, 0.75 mm ID; FHC, Bowdoinham, ME) using a horizontal puller (Sutter Instruments P-97; Novato, CA). Microelectrodes were filled with 3M potassium acetate. Manual micropositioners (Model 1480; Siskiyou Inc., Grants Pass, OR) were used to impale individual neurons during experiments. Current was delivered to electrodes using a multi-channel programmable stimulator (STG 1004; Multi-Channel Systems; Reutlingen, Germany) and the signal was recorded using a bridge amplifier (BA-1S; NPI, Tamm, Germany) and digitally converted for analysis (Axoscope; Molecular Devices, Sunnyvale, CA).
The presynaptic lateral nociceptive (N) and pressure (P) cells and the postsynaptic longitudinal (L) motor neuron and anterior pagoda (AP) cell were identified based on their position with the ganglion (Figure 1), size, and characteristic electrophysiological properties (size and shape of action potential). L motor neuron identification could be confirmed by recording from the electrically coupled contralateral L motor neurons and observing synchronous activity . For experiments utilizing N-to-L and P-to-L synapse recordings, the ganglion was pinned dorsal side up so that the L motor neurons could be located on the dorsal side along with access to the lateral-most N- and P-cells. For N-to-AP and P-to-AP synapse recordings, the ganglion was pinned ventral side up. Following pre-test recordings of the excitatory postsynaptic potentials (EPSPs), the ganglion was superfused with 2AG for 15 minutes and then returned to normal saline. In vehicle control experiments, 2AG was replaced with saline containing 0.01% DMSO. After one hour, the EPSP was retested (post-test). Separate electrode impalements of the same presynaptic and postsynaptic neuron were made for pre- and post-test recordings. Chronic intracellular recordings of these neurons were not carried out because this results in progressive rundown of the EPSP within 10–15 mins most likely due to damage caused by movements of the tissue during the electrode impalement (there are muscle fibers and connective tissue present in the leech CNS). Input resistance was recorded at the pre- and post-test level and only consistent, stable recordings were included in the data analysis (see Results section). The peak EPSP amplitude was recorded every 10 seconds and calculated by averaging 5–10 EPSP (pre- or post-test) sweeps.
For N-to-AP characterization experiments, the ganglion was pinned ventral side up as the lateral N-cell and AP-cell were both located on the ventral side. Treatments for the set of characterization experiments (high Mg2+ saline, high divalent saline, and CNQX) were perfused for 10 to 15 minutes between pre-test and post-test recordings while controls were carried out in constant perfusion of normal leech saline. Coupling experiments were performed by injecting single 500 msec hyperpolarizing current pulses with increasing amounts of current into the AP- or N-cell and recording any subsequent response from the opposite cell.
The effects of GABA on the N- and P-cells were tested using a “puffer” electrode (a patch electrode with a resistance of ≈ 5 MΩ) connected to a picospritzer. The puffer electrode was positioned approximately 100 μm from the N or P soma to prevent movement artifact and a 200 msec pulse of GABA was applied at 10 psi. The membrane potential of the P-cell was maintained at −50 mV. The membrane potential of the N-cell was maintained at −60 mV in order to prevent the cell from firing when GABA was applied. The concentration of GABA within the puffer electrode was 1 M, consistent with similar studies in the leech by Sargent et al. . This high concentration in the electrode was required due to the substantial amount of dilution that occurred by the time the expelled GABA reached the neuropil, which is relatively deep in the ganglion. That responses to GABA appear to arise from N- and P-cell processes in the neuropil is based on the delay between the GABA puff and the P- or N-cell response which ranged between 100–300 msec.
Post-test EPSP amplitudes and input resistance measurements were normalized relative to pre-test levels and presented as mean ± standard error. Statistical analyses using both independent and paired t-test or one-way analysis of variance (ANOVA) were performed to determine main effects with Newman-Keuls post-hoc tests to confirm the ANOVA results.
The authors thank the three anonymous reviewers whose comments significantly strengthened this manuscript. Supported by the National Science Foundation (IOS-1051734; BDB) and the National Institute of Neurological Disorders and Stroke (F31 NS074473; SY). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institute of Neurological Disorders And Stroke, the National Institutes of Health, and the National Science Foundation. Also supported by the University of South Dakota Council of Undergraduate Research and Creative Activities (CURCA) U.Discover Summer research fellowship (AH) and the Medical Student Summer Research program at the USD Sanford School of Medicine (AH).
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