Characterization of a Fatty Acid Amide Hydrolase (FAAH) in Hirudo verbana.

The endocannabinoid system plays a critical role in modulating both peripheral and central nervous system function. Despite being present throughout the animal kingdom, there has been relatively little investigation of the endocannabinoid system beyond the traditional animal model systems. In this study, we report on the identification and characterization of a fatty acid aminohydrolase (FAAH) in the medicinal leech, Hirudo verbana. FAAH is the primary enzyme responsible for metabolizing the endocannabinoid signaling molecule arachidonoyl ethanolamide (anandamide or AEA) and therefore plays a critical role in regulating AEA levels in the nervous system. This Hirudo FAAH (HirFAAH) is expressed in the leech central nervous system (CNS) and is an orthologue of FAAH-2 observed in vertebrates. Functionally, HirFAAH has serine hydrolase activity based on activity-based protein profiling (ABPP) studies using the fluorophosphonate probe TAMRA-FP. HirFAAH also hydrolyzes arachidonyl 7-amino, 4-methyl coumarin amide (AAMCA), a substrate specific to FAAH. Hydrolase activity during both the ABPP and AAMCA assays was eliminated by mutation at a conserved activity-binding site. Activity was also blocked by the known FAAH inhibitor, URB597. Treatment of Hirudo ganglia with URB597 potentiated synapses made by the pressure-sensitive mechanosensory neuron (P cell), mimicking the effects of exogenously applied AEA. The Hirudo CNS has been a useful system in which to study properties of endocannabinoid modulation of nociception relevant to vertebrates. Therefore, this characterization of HirFAAH is an important contribution to comparative studies of the endocannabinoid system.

Repetitive nociceptive stimulation elicits complex behavioral changes in Hirudo: evidence of arousal and motivational adaptations.

Appropriate responses to real or potential damaging stimuli to the body (nociception) are critical to an animal's short- and long-term survival. The initial goal of this study was to examine habituation of withdrawal reflexes (whole-body and local shortening) to repeated mechanical nociceptive stimuli (needle pokes) in the medicinal leech, Hirudo verbana, and assess whether injury altered habituation to these nociceptive stimuli. While repeated needle pokes did reduce shortening in H. verbana, a second set of behavior changes was observed. Specifically, animals began to evade subsequent stimuli by either hiding their posterior sucker underneath adjacent body segments or engaging in locomotion (crawling). Animals differed in terms of how quickly they adopted evasion behaviors during repeated stimulation, exhibiting a multi-modal distribution for early, intermediate and late evaders. Prior injury had a profound effect on this transition, decreasing the time frame in which animals began to carry out evasion and increasing the magnitude of these evasion behaviors (more locomotory evasion). The data indicate the presence in Hirudo of a complex and adaptive defensive arousal process to avoid noxious stimuli that is influenced by differences in internal states, prior experience with injury of the stimulated areas, and possibly learning-based processes.

Heterosynaptic long-term potentiation of non-nociceptive synapses requires endocannabinoids, NMDARs, CamKII, and PKCζ.

Noxious stimuli or injury can trigger long-lasting sensitization to non-nociceptive stimuli (referred to as allodynia in mammals). Long-term potentiation (LTP) of nociceptive synapses has been shown to contribute to nociceptive sensitization (hyperalgesia) and there is even evidence of heterosynaptic spread of LTP contributing to this type of sensitization. This study will focus on how activation of nociceptors elicits heterosynaptic LTP (hetLTP) in non-nociceptive synapses. Previous studies in the medicinal leech (Hirudo verbana) have demonstrated that high-frequency stimulation (HFS) of nociceptors produces both homosynaptic LTP as well as hetLTP in non-nociceptive afferent synapses. This hetLTP involves endocannabinoid-mediated disinhibition of non-nociceptive synapses at the presynaptic level, but it is not clear if there are additional processes contributing to this synaptic potentiation. In this study, we found evidence for the involvement of postsynaptic level change and observed that postsynaptic N-methyl-d-aspartate (NMDA) receptors (NMDARs) were required for this potentiation. Next, Hirudo orthologs for known LTP signaling proteins, CamKII and PKCζ, were identified based on sequences from humans, mice, and the marine mollusk Aplysia. In electrophysiological experiments, inhibitors of CamKII (AIP) and PKCζ (ZIP) were found to interfere with hetLTP. Interestingly, CamKII was found to be necessary for both induction and maintenance of hetLTP, whereas PKCζ was only necessary for maintenance. These findings show that activation of nociceptors can elicit a potentiation of non-nociceptive synapses through a process that involves both endocannabinoid-mediated disinhibition and NMDAR-initiated signaling pathways.NEW & NOTEWORTHY Pain-related sensitization involves increases in signaling by non-nociceptive sensory neurons. This can allow non-nociceptive afferents to have access to nociceptive circuitry. In this study, we examine a form of synaptic potentiation in which nociceptor activity elicits increases in non-nociceptive synapses. This process involves endocannabinoids, "gating" the activation of NMDA receptors, which in turn activate CamKII and PKCζ. This study provides an important link in how nociceptive stimuli can enhance non-nociceptive signaling related to pain.

Serotonin mediates stress-like effects on responses to non-nociceptive stimuli in the medicinal leech Hirudo verbana.

Noxious stimuli can elicit stress in animals that produce a variety of adaptations including changes in responses to nociceptive and non-nociceptive sensory input. One example is stress-induced analgesia that may be mediated, in part, by the endocannabinoid system. However, endocannabinoids can also have pro-nociceptive effects. In this study, the effects of electroshock, one experimental approach for producing acute stress, were examined on responses to non-nociceptive mechanical stimuli and nociceptive thermal stimuli in the medicinal leech (Hirudo verbana). The electroshock stimuli did not alter the leeches' responses to nociceptive stimuli, but did cause sensitization to non-nociceptive stimuli, characterized by a reduction in response threshold. These experiments were repeated with drugs that either blocked synthesis of the endocannabinoid transmitter 2-arachidonoylglycerol (2-AG) or transient receptor potential vanilloid (TRPV) channel, which is known to act as an endocannabinoid receptor. Surprisingly, neither treatment had any effect on responses following electroshock. However, the electroshock stimuli reliably increased serotonin (5-hydroxytryptamine or 5HT) levels in the H. verbana CNS. Injection of 5HT mimicked the effects of the electroshocks, sensitizing responses to non-nociceptive stimuli and having no effect on responses to nociceptive stimuli. Injections of the 5HT receptor antagonist methysergide reduced the sensitization effect to non-nociceptive stimuli after electroshock treatment. These results indicate that electroshocks enhance response to non-nociceptive stimuli but do not alter responses to nociceptive stimuli. Furthermore, while 5HT appears to play a critical role in this shock-induced sensitizing effect, the endocannabinoid system seems to have no effect.

Activity-Dependent Modulation of Tonic GABA Currents by Endocannabinoids in Hirudo verbana.

Endocannabinoids are lipid neuromodulators that are synthesized on demand and primarily signal in a retrograde manner to elicit depression of excitatory and inhibitory synapses. Despite the considerable interest in their potential analgesic effects, there is evidence that endocannabinoids can have both pro-nociceptive and anti-nociceptive effects. The mechanisms contributing to the opposing effects of endocannabinoids in nociception need to be better understood before cannabinoid-based therapies can be effectively utilized to treat pain. Using the medicinal leech, Hirudo verbana, this work investigates whether endocannabinoids modulate tonic inhibition onto non-nociceptive afferents. In voltage clamp recordings, we analyzed changes in the tonic inhibition in pressure-sensitive (P) cells following pre-treatment with endocannabinoids, 2-arachidonoylglycerol (2-AG) or anandamide (AEA). We also tested whether high frequency stimulation (HFS) of nociceptive (N) cells could also modulate tonic inhibition. Both endocannabinoid application and N cell HFS depressed tonic inhibition in the P cell. Depression of tonic inhibition by N cell HFS was blocked by SB 366791 (a TRPV1 inhibitor). SB 366791 also prevented 2-AG-and AEA-induced depression of tonic inhibition. HFS-induced depression was not blocked by tetrahydrolipstatin (THL), which prevents 2-AG synthesis, nor AM 251 (a CB1 receptor inverse agonist). These results illustrate a novel activity-dependent modulation of tonic GABA currents that is mediated by endocannabinoid signaling and is likely to play an important role in sensitization of non-nociceptive afferent pathways.

Approaches to studying injury-induced sensitization and the potential role of an endocannabinoid transmitter.

Endocannabinoids are traditionally thought to have an analgesic effect. However, it has been shown that while endocannabinoids can depress nociceptive signaling, they can also enhance non-nociceptive signaling. Therefore, endocannabinoids have the potential to contribute to non-nociceptive sensitization after an injury. Using Hirudo verbana (the medicinal leech), a model of injury-induced sensitization was developed in which a reproducible piercing injury was delivered to the posterior sucker of Hirudo. Injury-induced changes in the non-nociceptive threshold of Hirudo were determined through testing with Von Frey filaments and changes in the response to nociceptive stimuli were tested by measuring the latency to withdraw to a nociceptive thermal stimulus (Hargreaves apparatus). To test the potential role of endocannabinoids in mediating injury-induced sensitization, animals were injected with tetrahydrolipstatin (THL), which inhibits synthesis of the endocannabinoid transmitter 2-arachidonoylglycerol (2-AG). Following injury, a significant decrease in the non-nociceptive response threshold (consistent with non-nociceptive sensitization) and a significant decrease in the response latency to nociceptive stimulation (consistent with nociceptive sensitization) were observed. In animals injected with THL, a decrease in non-nociceptive sensitization in injured animals was observed, but no effect on nociceptive sensitization was observed.

Characterization of a monoacylglycerol lipase in the medicinal leech, Hirudo verbana.

Endocannabinoids are a class of lipid neuromodulators found throughout the animal kingdom. Among the endocannabinoids, 2-arachydonoyl glycerol (2-AG) is the most prevalent endocannabinoid and monoacylglycerol lipase (MAGL) is a serine hydrolase primarily responsible for metabolizing 2-AG in mammals. In the medicinal leech, Hirudo verbana, 2-AG has been found to be an important and multi-functional modulator of synaptic transmission and behavior. However, very little is known about the molecular components of its synthesis and degradation. In this study we have identified cDNA in Hirudo that encodes a putative MAGL (HirMAGL). The encoded protein exhibits considerable sequence and structural conservation with mammalian forms of MAGL, especially in the catalytic triad that mediates 2-AG metabolism. Additionally, HirMAGL transcripts are detected in the Hirudo central nervous system. When expressed in HEK 293 cells HirMAGL segregates to the plasma membrane as expected. It also exhibits serine hydrolase activity that is blocked when a critical active site residue is mutated. HirMAGL also demonstrates the capacity to metabolize 2-AG and this capacity is also prevented when the active site is mutated. Finally, HirMAGL activity is inhibited by JZL184 and MJN110, specific inhibitors of mammalian MAGL. To our knowledge these findings represent the first characterization of an invertebrate form of MAGL and show that HirMAGL exhibits many of the same properties as mammalian MAGL's that are responsible for 2-AG metabolism.

Comparative studies of endocannabinoid modulation of pain.

Cannabinoid-based therapies have long been used to treat pain, but there remain questions about their actual mechanisms and efficacy. From an evolutionary perspective, the cannabinoid system would appear to be highly conserved given that the most prevalent endogenous cannabinoid (endocannabinoid) transmitters, 2-arachidonyl glycerol and anandamide, have been found throughout the animal kingdom, at least in the species that have been analysed to date. This review will first examine recent findings regarding the potential conservation across invertebrates and chordates of the enzymes responsible for endocannabinoid synthesis and degradation and the receptors that these transmitters act on. Next, comparisons of how endocannabinoids modulate nociception will be examined for commonalities between vertebrates and invertebrates, with a focus on the medicinal leech Hirudo verbana. Evidence is presented that there are distinct, evolutionarily conserved anti-nociceptive and pro-nociceptive effects. The combined studies across various animal phyla demonstrate the utility of using comparative approaches to understand conserved mechanisms for modulating nociception. This article is part of the Theo Murphy meeting issue 'Evolution of mechanisms and behaviour important for pain'.

Interaction between NMDA Receptor- and Endocannabinoid-Mediated Modulation of Nociceptive Synapses.

Nociceptors, sensory neurons that detect damage or potential damage to the body, are the first stage of communicating noxious stimuli from the periphery to central nervous system (CNS). In this study, long-term potentiation (LTP) in the CNS of the medicinal leech, Hirudo verbana, was examined, taking advantage of the ability to selectively record from nociceptive synapses in this model organism. High frequency stimulation (HFS) of nociceptors produced a persistent increase in synaptic transmission and this LTP was both NMDA receptor-mediated and synapse-specific. Surprisingly, inhibition of NMDA receptors during HFS "uncovered" a persistent form of depression. This long-term depression (LTD) was mediated by the endocannabinoid 2-arachidonoyl glycerol (2-AG) acting on a TRPV (transient receptor potential vanilloid) -like channel. These observations suggest that (1) NMDA receptor mediated LTP is observed in nociceptors across both vertebrate and invertebrate phyla and (2) there may be an interaction between NMDA receptor-mediated and endocannabinoid-mediated forms of synaptic plasticity in nociceptors. Specifically, the NMDA receptor mediated processes may suppress endocannabinoid signaling. Such findings could be significant for understanding cellular mechanisms behind nociceptive sensitization and perhaps their contribution to chronic pain.

Are the persistent effects of "gate control" stimulation on nociception a form of generalization of habituation that is endocannabinoid-dependent?

Repetitive activation of non-nociceptive afferents is known to attenuate nociceptive signaling. However, the functional details of how this modulatory process operates are not understood and this has been a barrier in using such stimuli to effectively treat chronic pain. The present study tests the hypothesis that the ability of repeated non-nociceptive stimuli to reduce nociception is a form of generalized habituation from the non-nociceptive stimulus-response pathway to the nociceptive pathway. Habituation training, using non-nociceptive mechanosensory stimuli, did reduce responses to nociceptive thermal stimulation. This generalization of habituation to nociceptive stimuli required endocannabinoid-mediated neuromodulation, although disrupting of endocannabinoid signaling did not affect "direct" habituation of to the non-nociceptive stimulus. Surprisingly, the reduced response to nociceptive stimuli following habituation training was very long-lasting (3-8 days). This long-term habituation required endocannabinoid signaling during the training/acquisition phase, but endocannabinoids were not required for post-training retention phase. The implications of these results are that applying principles of habituation learning could potentially improve anti-nociceptive therapies utilizing repeated non-nociceptive stimulation such as transcutaneous nerve stimulation (TENS), spinal cord stimulation (SCS), or electro-acupuncture.

Endocannabinoid-mediated potentiation of nonnociceptive synapses contributes to behavioral sensitization.

Endocannabinoids, such as 2-arachidonoyl glycerol (2-AG) and anandamide, can elicit long-term depression of both excitatory and inhibitory synapses. This latter effect will result in disinhibition and would therefore be expected to produce an increase in neural circuit output. However, there have been no examples directly linking endocannabinoid-mediated disinhibition to a change in a functional neurobehavioral circuit. The present study uses the well-characterized central nervous system of the medicinal leech, Hirudo verbana, to examine the functional/behavioral relevance of endocannabinoid modulation of an identified afferent synapse. Bath application of 2-AG potentiates synaptic transmission by pressure-sensitive sensory neurons (P cells) as well as the magnitude of the defensive shortening reflex elicited by P-cell stimulation. This potentiation requires activation of TRPV-like channels. Endocannabinoid/TRPV signaling was found to produce sensitization of the shortening reflex elicited by either direct stimulation of nearby nociceptive afferents (N cells) or noxious stimulation applied to skin several segments away. In both cases, heterosynaptic potentiation of P-cell synapses was observed in parallel with an increase in the magnitude of elicited shortening and both synaptic and behavioral effects were blocked by pharmacological inhibition of 2-AG synthesis or TRPV-like channel activation. Serotonin (5-HT) is known to play a critical role in sensitization in Hirudo and other animals, and the 5-HT2 receptor antagonist ritanserin also blocked behavioral sensitization and the accompanying synaptic potentiation. These findings suggest a novel, endocannabinoid-mediated contribution to behavioral sensitization that may interact with known 5-HT-dependent modulatory processes. NEW & NOTEWORTHY There is considerable interest in the analgesic potential of cannabinoids. However, there is evidence that the cannabinoid system can have both pro- and antinociceptive effects. This study examines how an endogenous cannabinoid transmitter can potentiate nonnociceptive synapses and enhance their capacity to elicit a nocifensive behavioral response.

Comparative biology of pain: What invertebrates can tell us about how nociception works.

The inability to adequately treat chronic pain is a worldwide health care crisis. Pain has both an emotional and a sensory component, and this latter component, nociception, refers specifically to the detection of damaging or potentially damaging stimuli. Nociception represents a critical interaction between an animal and its environment and exhibits considerable evolutionary conservation across species. Using comparative approaches to understand the basic biology of nociception could promote the development of novel therapeutic strategies to treat pain, and studies of nociception in invertebrates can provide especially useful insights toward this goal. Both vertebrates and invertebrates exhibit segregated sensory pathways for nociceptive and nonnociceptive information, injury-induced sensitization to nociceptive and nonnociceptive stimuli, and even similar antinociceptive modulatory processes. In a number of invertebrate species, the central nervous system is understood in considerable detail, and it is often possible to record from and/or manipulate single identifiable neurons through either molecular genetic or physiological approaches. Invertebrates also provide an opportunity to study nociception in an ethologically relevant context that can provide novel insights into the nature of how injury-inducing stimuli produce persistent changes in behavior. Despite these advantages, invertebrates have been underutilized in nociception research. In this review, findings from invertebrate nociception studies are summarized, and proposals for how research using invertebrates can address questions about the fundamental mechanisms of nociception are presented.

Differences in chloride gradients allow for three distinct types of synaptic modulation by endocannabinoids.

Endocannabinoids can elicit persistent depression of excitatory and inhibitory synapses, reducing or enhancing (disinhibiting) neural circuit output, respectively. In this study, we examined whether differences in Cl(-) gradients can regulate which synapses undergo endocannabinoid-mediated synaptic depression vs. disinhibition using the well-characterized central nervous system (CNS) of the medicinal leech, Hirudo verbana Exogenous application of endocannabinoids or capsaicin elicits potentiation of pressure (P) cell synapses and depression of both polymodal (Npoly) and mechanical (Nmech) nociceptive synapses. In P synapses, blocking Cl(-) export prevented endocannabinoid-mediated potentiation, consistent with a disinhibition process that has been indicated by previous experiments. In Nmech neurons, which are depolarized by GABA due to an elevated Cl(-) equilibrium potentials (ECl), endocannabinoid-mediated depression was prevented by blocking Cl(-) import, indicating that this decrease in synaptic signaling was due to depression of excitatory GABAergic input (disexcitation). Npoly neurons are also depolarized by GABA, but endocannabinoids elicit depression in these synapses directly and were only weakly affected by disruption of Cl(-) import. Consequently, the primary role of elevated ECl may be to protect Npoly synapses from disinhibition. All forms of endocannabinoid-mediated plasticity required activation of transient potential receptor vanilloid (TRPV) channels. Endocannabinoid/TRPV-dependent synaptic plasticity could also be elicited by distinct patterns of afferent stimulation with low-frequency stimulation (LFS) eliciting endocannabinoid-mediated depression of Npoly synapses and high-frequency stimulus (HFS) eliciting endocannabinoid-mediated potentiation of P synapses and depression of Nmech synapses. These findings demonstrate a critical role of differences in Cl(-) gradients between neurons in determining the sign, potentiation vs. depression, of synaptic modulation under normal physiological conditions.

Physiological, pharmacological and behavioral evidence for a TRPA1 channel that can elicit defensive responses in the medicinal leech.

Transient receptor potential ankyrin subtype 1 (TRPA1) channels are chemosensitive to compounds such as allyl isothiocyanate (AITC, the active component of mustard oil) and other reactive electrophiles and may also be thermodetectors in many animal phyla. In this study, we provide the first pharmacological evidence of a putative TRPA1-like channel in the medicinal leech. The leech's polymodal nociceptive neuron was activated by both peripheral and central application of the TRPA1 agonist AITC in a concentration-dependent manner. Responses to AITC were inhibited by the selective TRPA1 antagonist HC030031, but also by the TRPV1 antagonist SB366791. Other TRPA1 activators - N-methylmaleimide (NMM) and cinnamaldehyde (CIN) - also activated this nociceptive neuron, although HC030031 only inhibited the effects of NMM. The polymodal nociceptive neurons responded to moderately cold thermal stimuli (<17°C) and these responses were blocked by HC030031. AITC sensitivity was also found in the pressure-sensitive sensory neurons and was blocked by HC030031, but not by SB366791. AITC elicited a nocifensive withdrawal of the posterior sucker in a concentration-dependent manner that could be attenuated with HC030031. Peripheral application of AITC in vivo also produced swimming-like behavior that was attenuated by HC030031. These results suggest the presence of a TRPA1-like channel in the medicinal leech nervous system that responds to cold temperatures and may interact with the leech TRPV-like channel.

Physiological and behavioral evidence of a capsaicin-sensitive TRPV-like channel in the medicinal leech.

Transient receptor potential vanilloid (TRPV) channels are found throughout the animal kingdom, where they play an important role in sensory transduction. In this study, we combined physiological studies with in vivo behavioral experiments to examine the presence of a putative TRPV-like receptor in the medicinal leech, building upon earlier studies in this lophotrochozoan invertebrate. The leech polymodal nociceptive neuron was activated by both peripheral and central application of the TRPV1-activator capsaicin in a concentration-dependent manner, with 100 µmol l(-1) being the lowest effective concentration. Responses to capsaicin were inhibited by the selective TRPV1 antagonist SB366791. The polymodal nociceptive neuron also responded to noxious thermal stimuli (>40°C), and this response was also blocked by SB366791. Capsaicin sensitivity was selective to the polymodal nociceptor with no direct response being elicited in the mechanical nociceptive neuron or in the non-nociceptive touch- or pressure-sensitive neurons. Capsaicin also elicited nocifensive behavioral responses (withdrawals and locomotion) in a concentration-dependent manner, and these behavioral responses were significantly attenuated with SB366791. These results suggest the presence of a capsaicin-sensitive TRPV-like channel in the medicinal leech central nervous system and are relevant to the evolution of nociceptive signaling.

Nonnociceptive afferent activity depresses nocifensive behavior and nociceptive synapses via an endocannabinoid-dependent mechanism.

Previously, low-frequency stimulation (LFS) of a nonnociceptive touch-sensitive neuron has been found to elicit endocannabinoid-dependent long-term depression (eCB-LTD) in nociceptive synapses in the leech central nervous system (CNS) that requires activation of a presynaptic transient receptor potential vanilloid (TRPV)-like receptor by postsynaptically synthesized 2-arachidonoyl glycerol (2-AG). This capacity of nonnociceptive afferent activity to reduce nociceptive signaling resembles gate control of pain, albeit longer lasting in these synaptic experiments. Since eCB-LTD has been observed at a single sensory-motor synapse, this study examines the functional relevance of this mechanism, specifically whether this form of synaptic plasticity has similar effects at the behavioral level in which additional, intersegmental neural circuits are engaged. Experiments were carried out using a semi-intact preparation that permitted both synaptic recordings and monitoring of the leech whole body shortening, a defensive withdrawal reflex that was elicited via intracellular stimulation of a single nociceptive neuron (the N cell). The same LFS of a nonnociceptive afferent that induced eCB-LTD in single synapses also produced an attenuation of the shortening reflex. Similar attenuation of behavior was also observed when 2-AG was applied. LFS-induced behavioral and synaptic depression was blocked by tetrahydrolipstatin (THL), a diacylglycerol lipase inhibitor, and by SB366791, a TRPV1 antagonist. The effects of both THL and SB366791 were observed following either bath application of the drug or intracellular injection into the presynaptic (SB366791) or postsynaptic (THL) neuron. These findings demonstrate a novel, endocannabinoid-based mechanism by which nonnociceptive afferent activity may modulate nocifensive behaviors via action on primary afferent synapses.

Differential modulation of nociceptive versus non-nociceptive synapses by endocannabinoids.

BACKGROUND: 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. RESULTS: 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. CONCLUSIONS: 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.

Endocannabinoid-dependent long-term depression in a nociceptive synapse requires coordinated presynaptic and postsynaptic transcription and translation.

Endocannabinoids (eCBs) play an important role in long-term regulation of synaptic signaling in both vertebrates and invertebrates. In this study, the role of transcription- and translation-dependent processes in presynaptic versus postsynaptic neurons was examined during eCB-mediated synaptic plasticity in the CNS of the leech. Low-frequency stimulation (LFS) of non-nociceptive afferents elicits eCB-dependent long-term depression (eCB-LTD) heterosynaptically in nociceptive synapses that lasts at least 2 h. Bath application of emetine, a protein synthesis inhibitor, blocked eCB-LTD after afferent LFS or exogenous eCB application, indicating that this depression was translation dependent. Bath application of actinomycin D, an irreversible RNA synthesis inhibitor, or 5,6-dichlorobenzimidazole 1-ß-d-ribofurandoside (DRB), a reversible RNA synthesis inhibitor, also prevented eCB-LTD. Selective injection of DRB or emetine into the presynaptic or postsynaptic neuron before LFS indicated that eCB-LTD required transcription and translation in the postsynaptic neuron but only translation in the presynaptic cell. Depression observed immediately after LFS was also blocked when these transcription- and translation-dependent processes were inhibited. It is proposed that induction of eCB-LTD in this nociceptive synapse requires the coordination of presynaptic protein synthesis and postsynaptic mRNA and protein synthesis. These findings provide significant insights into both eCB-based synaptic plasticity and understanding how activity in non-nociceptive afferents modulates nociceptive pathways.

Long-term depression of nociceptive synapses by non-nociceptive afferent activity: role of endocannabinoids, Ca²+, and calcineurin.

Activity in non-nociceptive afferents is known to produce long-lasting decreases in nociceptive signaling, often referred to as gate control, but the cellular mechanisms mediating this form of neuroplasticity are poorly understood. In the leech, activation of non-nociceptive touch (T) mechanosensory neurons induces a heterosynaptic depression of nociceptive (N) synapses that is endocannabinoid-dependent. This heterosynaptic, endocannabinoid-dependent long-term depression (ecLTD) is observed where the T- and N-cells converge on a common postsynaptic target, in this case the motor neuron that innervates the longitudinal muscles (L-cells) that contributes to a defensive withdrawal reflex. Depression in the nociceptive synapse required both presynaptic and postsynaptic increases in intracellular Ca²âº. Activation of the Ca²âº-sensitive protein phosphatase calcineurin was also required, but only in the presynaptic neuron. Heterosynaptic ecLTD was unaffected by antagonists for NMDA or metabotropic glutamate receptors, but was blocked by the 5-HT2 receptor antagonist ritanserin. Depression was also blocked by the CB1 receptor antagonist rimonabant, but this is thought to represent an effect on a TRPV-like receptor. This heterosynaptic, endocannabinoid-dependent modulation of nociceptive synapses represents a novel mechanism for regulating how injury-inducing or painful stimuli are transmitted to the rest of the central nervous system.