Properties of cannabinoid-dependent long-term depression in the leech

Previously, a cannabinoid-dependent form of long-term depression (LTD) was discovered at the polysynaptic connection between the touch mechanosensory neuron and the S interneuron (Li and Burrell in J Comp Physiol A 195:831–841, 2009). In the present study, the physiological properties of this cannabinoid-dependent LTD were examined. Increases in intracellular calcium in the S interneuron are necessary for this form of LTD in this circuit. Calcium signals contributing to cannabinoid-dependent LTD are mediated by voltage-dependent calcium channel and release of calcium from intracellular stores. Inositol triphosphate receptors, but not ryanodine receptors, appear to mediate this store-released calcium signal. Cannabinoid-dependent LTD also requires activation of metabotropic serotonin receptors, possibly a serotonin type 2-like receptor. Finally, this form of LTD involves the stimulation of nitric oxide synthase and a decrease in cyclic adenosine monophosphate signaling, both of which appeared to be downstream of cannabinoid receptor activation. Based on these findings, the cellular signaling mechanisms of cannabinoid-dependent LTD in the leech are remarkably similar to vertebrate forms of cannabinoid-dependent synaptic plasticity.     

Endocannabinoids control the induction of cerebellar LTD

The long-term depression (LTD) of parallel fiber (PF) synapses onto Purkinje cells plays a central role in motor learning. Endocannabinoid release and LTD induction both depend upon activation of the metabotropic glutamate receptor mGluR1, require postsynaptic calcium increases, are synapse specific, and have a similar dependence on the associative activation of PF and climbing fiber synapses. These similarities suggest that endocannabinoid release could account for many features of cerebellar LTD. Here we show that LTD induction is blocked by a cannabinoid receptor (CB1R) antagonist, by inhibiting the synthesis of the endocannabinoid 2-arachidonyl glycerol (2-AG), and is absent in mice lacking the CB1R. Although CB1Rs are prominently expressed presynaptically at PF synapses, LTD is expressed postsynaptically. In contrast, a previously described transient form of inhibition mediated by endocannabinoids is expressed presynaptically. This indicates that Purkinje cells release 2-AG that activates CB1Rs to both transiently inhibit release and induce a postsynaptic form of LTD.     

Corticospinal transmission to motoneurons in cervical spinal cord slices from adult rats

Cervical spinal cord slices were prepared from adult rats. Intracellular recordings from motoneurons revealed that electrical stimulation of the ventralmost part of the dorsal funiculus (which contains primarily descending corticospinal axons) elicited EPSPs in 75% of the neurons. The latencies of these EPSPs tended to be shorter than those elicited by dorsal horn gray matter stimulation. Pairs of subthreshold dorsal funiculus stimuli were able to elicit action potentials in motoneurons. These data are consistent with previous morphological and electrophysiological studies indicating that cervical motoneurons receive both mono-and polysynaptic corticospinal inputs. In addition, motoneurons were markedly depolarized by iontophoretic application of AMPA or KA (7 out of 7 neurons), but only weakly depolarized by NMDA (1 out of 6 neurons). CNQX (but not AP-5) blocked EPSPs elicited by dorsal funiculus stimulation. Thus, corticospinal transmission to motoneurons is mediated primarily by non-NMDA glutamate receptors.     

Activation of metabotropic glutamate receptors in conjunction with postsynaptic depolarization triggers a long-term depression of the N-methyl-D-aspartate receptor-mediated synaptic potential in the rat hippocampus

The mechanism responsible for long-term depression (LTD) of pharmacologically isolated N-methyl-D-aspartate (NMDA) receptor-mediated excitatory postsynaptic potential (EPSP_NMDA) was studied. Intracellular recordings were made from CA1 cells of rat hippocampal slices in the presence of 6-cyano-7-nitroquinoxaline-2,3-dione (10 µM) and picrotoxin (50 µM), which block non-NMDA and GABA_A receptors, respectively. Intracellular injections of depolarizing pulses (500 ms, 0.3–0.7 nA) at 1 Hz for 5 min in the absence of synaptic stimulation caused a persistent increase in the amplitude of EPSP_NMDA. However, coupling postsynaptic depolarization with synaptic activity induced LTD. The EPSP_NMDA LTD could be blocked byL-2-amino-3-phosphonopropionic acid (50 µM) or (RS)--methyl-4-carboxyphenylglycine (200 µM), specific antagonists for metabotropic glutamate receptors (mGluR). Furthermore, application oftrans-1-aminocyclopentane-1,3-dicarboxylic acid (t-ACPD, 50 µM), a specific mGluR agonist, in conjunction with postsynaptic depolarizing elicited LTD. In contrast, the mGluR agonists quisqualate or t-ACPD when given alone produced a sustained enhancement of EPSP_NMDA. Finally, coupled depolarization did not evoke LTD in slices pretreated with the protein kinase C (PKC) inhibitor calphostin c (60 nM). The present results demonstrate that activation of mGluR is necessary for the induction of LTD of EPSP_NMDA and suggest that NMDA receptors are subject to bidirectional regulation by mGluR. Furthermore, the induction of LTD is likely to involve the stimulation of PKC.     

Insulin modulates hippocampal activity-dependent synaptic plasticity in a N-methyl-d-aspartate receptor and phosphatidyl-inositol-3-kinase-dependent manner

Insulin and its receptor are both present in the central nervous system and are implicated in neuronal survival and hippocampal synaptic plasticity. Here we show that insulin activates phosphatidylinositol 3-kinase (PI3K) and protein kinase B (PKB), and results in an induction of long-term depression (LTD) in hippocampal CA1 neurones. Evaluation of the frequency-response curve of synaptic plasticity revealed that insulin induced LTD at 0.033 Hz and LTP at 10 Hz, whereas in the absence of insulin, 1 Hz induced LTD and 100 Hz induced LTP. LTD induction in the presence of insulin required low frequency synaptic stimulation (0.033 Hz) and blockade of GABAergic transmission. The LTD or LTP induced in the presence of insulin was N-methyl-d-aspartate (NMDA) receptor specific as it could be inhibited by alpha-amino-5-phosphonopentanoic acid (APV), a specific NMDA receptor antagonist. LTD induction was also facilitated by lowering the extracellular Mg(2+) concentration, indicating an involvement of NMDA receptors. Inhibition of PI3K signalling or discontinuing synaptic stimulation also prevented this LTD. These results show that insulin modulates activity-dependent synaptic plasticity, which requires activation of NMDA receptors and the PI3K pathway. The results obtained provide a mechanistic link between insulin and synaptic plasticity, and explain how insulin functions as a neuromodulator.     

Glutamate as a transmitter in the sensory pathway from prostomial lip to serotonergic Retzius neurons in the medicinal leechHirudo

The involvement of glutamate in putative ingestive sensory pathways affecting the excitability of serotonergic Retzius neurons (RZ) in the leech CNS was investigated with a pharmacological approach. Exposure of the prostomial lip to 150mm NaCl and 1mm arginine produced excitatory as well as inhibitory responses in RZ found in the reproductive segments, while only excitatory responses were elicted in standard midbody RZ. Antagonists of glutamatergic receptors of the kainate/quisqualate type effectively inhibited chemosensory dependent excitation of RZ. Antagonists of glutamatergic receptors of theN-methyld-aspartate type were ineffective in this regard. Cephalic nerve stimulation, like chemical stimulation of the lip, produced segment-specific responses in midbody RZ. Both the polysynaptic and monosynaptic components of the excitatory response of standard midbody RZ following cephalic nerve stimulation were inhibited in the presence of the kainate/quisqualate antagonist DNQX. These data suggest a role for glutamate as a transmitter in the neural circuitry from receptors of the leech prostomial lip to serotonergic RZ.     

Single synaptic events evoke NMDA receptor-mediated release of calcium from internal stores in hippocampal dendritic spines

We have used confocal microscopy to monitor synaptically evoked Ca2+ transients in the dendritic spines of hippocampal pyramidal cells. Individual spines respond to single afferent stimuli (<0.1 Hz) with Ca2+ transients or failures, reflecting the probability of transmitter release at the activated synapse. Both AMPA and NMDA glutamate receptor antagonists block the synaptically evoked Ca2+ transients; the block by AMPA antagonists is relieved by low Mg2+. The Ca2+ transients are mainly due to the release of calcium from internal stores, since they are abolished by antagonists of calcium-induced calcium release (CICR); CICR antagonists, however, do not depress spine Ca2+ transients generated by backpropagating action potentials. These results have implications for synaptic plasticity, since they show that synaptic stimulation can activate NMDA receptors, evoking substantial Ca2+ release from the internal stores in spines without inducing long-term potentiation (LTP) or depression (LTD).     

Involvement of presynaptic N-methyl-D-aspartate receptors in cerebellar long-term depression.

At the cerebellar synapses between parallel fibers (PFs) and Purkinje cells (PCs), long-term depression (LTD) of the excitatory synaptic current has been assumed to be independent of the N-methyl-D-aspartate (NMDA) receptor activation because PCs lack NMDA receptors. However, we now report that LTD is suppressed by NMDA receptor antagonists that act on presynaptic NMDA receptors of the PFs. This effect is still observed when the input is restricted to a single fiber. Therefore, LTD does not require the spatial integration of multiple inputs. In contrast, it involves a temporal integration, since reliable LTD induction requires the PFs to fire two action potentials in close succession. This implies that LTD will selectively depress the response to a burst of presynaptic action potentials.     

Involvement of NMDA Receptor in the Modulation of Excitatory and Inhibitory Amino Acid Neurotransmitters Release in Cortical Neurons

The purpose of this paper was to examine the function of N-methyl-D-aspartate (NMDA) glutamate receptor in cortical neurons on amino acid neurotransmitters release as well as the fraction of neurons implicated in the response of this receptor. Local stimulation of these cells at different concentrations of NMDA, agonist of this ionotropic glutamate receptor, produced a dose dependent release of aspartate, glutamate, glycine and GABA. These effects were blocked by DAP5, an antagonist of the NMDA receptor. The amino acid Ca²⁺ dependent release mediated by the NMDA receptor, is induced by the opening of voltage-dependent Ca²⁺ channels that this receptor promotes. Ca⁺⁺ movements were explored in single cells loaded with fura-2. When single cells were stimulated with 100 μM NMDA, the calcium recording performed showed that 82% of the cells responded to this agonist increasing the intracellular calcium concentration, although the amplitude of these increments was variable. The results suggest that NMDA-elicited neurotransmitter release from cortical neurons involves Ca²⁺-dependent and Ca²⁺-independent components, as well as neuron depolarisation, and different VDCC subtypes of N, P/Q or L depending of the amino acid neurotransmitter release elicited by this receptor.     

Anandamide, but not 2-arachidonoylglycerol, accumulates during in vivo neurodegeneration

Endogenous cannabinoid receptor ligands (endocannabinoids) may rescue neurons from glutamate excitotoxicity. As these substances also accumulate in cultured immature neurons following neuronal damage, elevated endocannabinoid concentrations may be interpreted as a putative neuroprotective response. However, it is not known how glutamatergic insults affect in vivo endocannabinoid homeostasis, i.e. N-arachidonoylethanolamine (anandamide) and 2-arachidonoylglycerol (2-AG), as well as other constituents of their lipid families, N-acylethanolamines (NAEs) and 2-monoacylglycerols (2-MAGs), respectively. Here we employed three in vivo neonatal rat models characterized by widespread neurodegeneration as a consequence of altered glutamatergic neurotransmission and assessed changes in endocannabinoid homeostasis. A 46-fold increase of cortical NAE concentrations (anandamide, 13-fold) was noted 24 h after intracerebral NMDA injection, while less severe insults triggered by mild concussive head trauma or NMDA receptor blockade produced a less pronounced NAE accumulation. By contrast, levels of 2-AG and other 2-MAGs were virtually unaffected by the insults employed, rendering it likely that key enzymes in biosynthetic pathways of the two different endocannabinoid structures are not equally associated to intracellular events that cause neuronal damage in vivo. Analysis of cannabinoid CB(1) receptor mRNA expression and binding capacity revealed that cortical subfields exhibited an up-regulation of these parameters following mild concussive head trauma and exposure to NMDA receptor blockade. This may suggest that mild to moderate brain injury may trigger elevated endocannabinoid activity via concomitant increase of anandamide levels, but not 2-AG, and CB(1) receptor density.     

Occurrence and possible biological role of the endocannabinoid system in the sea squirt Ciona intestinalis

A cannabinoid receptor orthologue (CiCBR) has been described in the sea squirt Ciona intestinalis. Here we report that CiCBR mRNA expression is highest in cerebral ganglion, branchial pharynx, heart and testis of C. intestinalis, and that this organism also contains cannabinoid receptor ligands and some of the enzymes for ligand biosynthesis and inactivation. Using liquid chromatography-mass spectrometry, the endocannabinoid anandamide was found in all tissues analysed (0.063-5.423 pmol/mg of lipid extract), with the highest concentrations being found in brain and heart. The endocannabinoid 2-arachidonoylglycerol (2-AG) was fivefold more abundant than anandamide, and was most abundant in stomach and intestine and least abundant in heart and ovaries (2.677-50.607 pmol/mg of lipid extract). Using phylogenomic analysis, we identified orthologues of several endocannabinoid synthesizing and degrading enzymes. In particular, we identified and partly sequenced a fatty acid amide hydrolase (FAAH) orthologue, showing 44% identity with human FAAH and containing nearly all the amino acids necessary for a functional FAAH enzyme. Ciona intestinalis also contained specific binding sites for cannabinoid receptor ligands, and an amidase enzyme with pH-dependency and subcellular/tissue distribution similar to mammalian FAAHs. Finally, a typical C. intestinalis behavioural response, siphon reopening after closure induced by mechanical stimulation, was inhibited by the cannabinoid receptor agonist HU-210, and this effect was significantly attenuated by mammalian cannabinoid receptor antagonists.     

Functional characteristics ofl-glutamate,N-methyl-d-aspartate and kainate receptors in isolated brain synaptic membranes

l-Glutamic acid (l-Glu) and other excitatory amino acids and amino acid analogs enhanced [³⁵S]thiocyanate (SCN^–) uptake in isolated-resealed synaptic membrane vesicles. The SCN^– uptake was used as a measure of membrane depolarization to evaluate the characteristics of functional excitatory amino acid receptors in the synaptic membranes.N-Methyl-d-aspartate (NMDA) andl-Glu produced additive effects on SCN^– accumulation indicating the presence of distinctl-Glu and NMDA receptors. On the other hand, kainic acid (KA) andl-Glu shared either common receptor sites or ion channels. The effects of antagonists on NMDA,l-Glu, and KA stimulation of SCN^– influx were consistent with previously reported electrophysiologic observations in intact neurons.     

Induction of Long-Term Depression of Synaptic Transmission in a Co-Culture of DRG and Spinal Dorsal Horn Neurons of Rats

In a co-culture of dissociated neurons of lumbar dorsal root ganglia (DRG) and spinal dorsal horn (DH) neurons of newborn rats, we examined peculiarities of induction of long-term depression (LTD) of synaptic transmission through synapses formed by primary afferents on DH neurons. Induction of LTD was provided by low-frequency (5 sec⁻¹) microstimulation of single DRG neurons. Ion currents were simultaneously recorded in pre- and post-synaptic cells using a dual whole-cell path-clamp technique. Parameters of evoked excitatory and inhibitory postsynaptic currents (eEPSCs and eIPSCs, respectively) initiated in DH neurons by intracellular stimulation of DRG neurons were analyzed. Monosynaptic eEPSC mediated by activation of AMPA receptors demonstrated no sensitivity to blockers of NMDA and kainate receptors (20 μM D_L-AP5 and 10 μM SIM 2081, respectively), but were entirely blocked upon applications of 10 μM DNQX. Monosynaptic glycinergic eIPSCs found in some of the DH neurons were blocked by 1 μM strychnine and were insensitive to 10 μM bicuculline and blockers of glutamatergic neurotransmission, D_L-AP5 and DNQX. Long-lasting (360 sec) low-frequency stimulation of DRG neurons did not affect the amplitude of glycineinduced eIPSCs in DH neurons. At the same time, such stimulation of DRG neurons evoked a drop in the amplitude of AMPA-activated eEPSCs in DH neurons to 41.6 ± 2.5%, on average, as compared with the analogous index in the control. This effect lasted at least 20 min after stimulation. Long-term depression of glutamatergic transmission in DH neurons was observed at the holding potential of −70 mV and did not change after applications of 10 μM bicuculline and 1 μM strychnine. The LTD intensity depended on the duration of low-frequency stimulation of primary afferent neurons. Sequential stimulation of DRG neurons lasting 120, 160, 200, and 240 sec resulted in decreases in the eEPSC amplitude in DH neurons to 85.6 ± 3.9, 62.7 ± 4.3, 51.8 ± 3.5, and 41.6 ±2.5% with respect to control values. Our findings show that use-dependent induction of homosynaptic LTD of glutamatergic transmission is possible at the level of a separate pair of synaptically connected DRG and DH neurons under co-culturing conditions. Such LTD of glutamatergic synaptic transmission mostly mediated by activation of AMPA receptors depends on the duration of activation of a presynaptic DRG neuron and does not need depolarization of a postsynaptic DH neuron.     

Inhibition of associative long-term depression by activation of β-adrenergic receptors in rat hippocampal CA1 synapses

The aim of this study was to investigate the role of β-adrenergic receptors in modulating associative long-term depression (LTD) at CA1 synapses in rat hippocampal slices. Standard extracellular electrophysiological techniques were employed to record field excitatory post-synaptic potential (fEPSP) activity and to induce associative LTD. Two independent Schaffer collateral pathways were elicited in hippocampal CA1 areas. In one (weak) pathway, the stimulating intensity was adjusted to elicit small fEPSP activity (20–30% of the maximum response). In contrast, 80–90% of the maximum response was evoked in the other (strong) pathway. Associative LTD of weak pathway could be induced by paired stimulation of weak and the strong pathways, repeated 100 times at 0.167 Hz. The associative LTD of weak pathway was NMDA receptor- and phophatase 2B dependent, because bath application of 50 μM D, L-AP5 or 10 μM cypermethrin blocked its induction. Bath application of 1 μM isoproterenol inhibited associative LTD, and this effect was blocked by timolol, suggesting the involvement of β-adrenergic receptors. The inhibitory effect of β-adrenergic receptors on LTD induction was blocked in slices pretreated with inhibitors of protein kinase A and mitogen-activated protein kinase, suggesting that these signal cascades are downstream effectors following activation of β-adrenergic receptors. Nevertheless, bath application of timolol or cypermethrin alone did not have significant effect on associative LTD induction, suggesting neither endogenous function of β-adrenergic receptor nor endogenous PKA activity does have a role in associative LTD induction.     

Biosynthesis and degradation of anandamide and 2-arachidonoylglycerol and their possible physiological significance

N -arachidonoylethanolamine (anandamide) was the first endogenous cannabinoid receptor ligand to be discovered. Dual synthetic pathways for anandamide have been proposed. One is the formation from free arachidonic acid and ethanolamine, and the other is the formation from N -arachidonoyl phosphatidylethanolamine (PE) through the action of a phosphodiesterase. These pathways, however, do not appear to be able to generate a large amount of anandamide, at least under physiological conditions. The generation of anandamide from free arachidonic acid and ethanolamine is catalyzed by a degrading enzyme anandamide amidohydrolase/fatty acid amide hydrolase operating in reverse and requires large amounts of substrates. As for the second pathway, arachidonic acids esterified at the 1-position of glycerophospholipids, which are mostly esterified at the 2-position, are utilized for the formation of N -arachidonoyl PE, a stored precursor form of anandamide. In fact, the actual levels of anandamide in various tissues are generally low except in a few cases. 2-Arachidonoylglycerol (2-AG) was the second endogenous cannabinoid receptor ligand to be discovered. 2-AG is a degradation product of arachidonic acid-containing glycerophospholipids such as inositol phospholipids. Several investigators have demonstrated that 2-AG is produced in a variety of tissues and cells upon stimulation. 2-AG acts as a full agonist at the cannabinoid receptors (CB1 and CB2). Evidence is gradually accumulating and indicates that 2-AG is the most efficacious endogenous natural ligand for the cannabinoid receptors.In this review, we summarize the tissue levels, biosynthesis, degradation and possible physiological significance of two endogenous cannabimimetic molecules, anandamide and 2-AG.     

The endocannabinoid 2-arachidonoyl-glycerol activates human neutrophils: critical role of its hydrolysis and de novo leukotriene B4 biosynthesis

Although endocannabinoids are important players in nociception and obesity, their roles as immunomodulators remain elusive. The main endocannabinoids described to date, namely 2-arachidonoyl-glycerol (2-AG) and arachidonyl-ethanolamide (AEA), induce an intriguing profile of pro- and anti-inflammatory effects. This could relate to cell-specific cannabinoid receptor expression and/or the action of endocannabinoid-derived metabolites. Importantly, 2-AG and AEA comprise a molecule of arachidonic acid (AA) in their structure and are hydrolyzed rapidly. We postulated the following: 1) the released AA from endocannabinoid hydrolysis would be metabolized into eicosanoids; and 2) these eicosanoids would mediate some of the effects of endocannabinoids. To confirm these hypotheses, experiments were performed in which freshly isolated human neutrophils were treated with endocannabinoids. Unlike AEA, 2-AG stimulated myeloperoxidase release, kinase activation, and calcium mobilization by neutrophils. Although 2-AG did not induce the migration of neutrophils, it induced the release of a migrating activity for neutrophils. 2-AG also rapidly (1 min) induced a robust biosynthesis of leukotrienes, similar to that observed with AA. The effects of 2-AG were not mimicked nor prevented by cannabinoid receptor agonists or antagonists, respectively. Finally, the blockade of either 2-AG hydrolysis, leukotriene (LT) B(4) biosynthesis, or LTB(4) receptor 1 activation prevented all the effects of 2-AG on neutrophil functions. In conclusion, we demonstrated that 2-AG potently activates human neutrophils. This is the consequence of 2-AG hydrolysis, de novo LTB(4) biosynthesis, and an autocrine activation loop involving LTB(4) receptor 1.     

The evolution and comparative neurobiology of endocannabinoid signalling

CB₁- and CB₂-type cannabinoid receptors mediate effects of the endocannabinoids 2-arachidonoylglycerol (2-AG) and anandamide in mammals. In canonical endocannabinoid-mediated synaptic plasticity, 2-AG is generated postsynaptically by diacylglycerol lipase alpha and acts via presynaptic CB₁-type cannabinoid receptors to inhibit neurotransmitter release. Electrophysiological studies on lampreys indicate that this retrograde signalling mechanism occurs throughout the vertebrates, whereas system-level studies point to conserved roles for endocannabinoid signalling in neural mechanisms of learning and control of locomotor activity and feeding. CB₁/CB₂-type receptors originated in a common ancestor of extant chordates, and in the sea squirt Ciona intestinalis a CB₁/CB₂-type receptor is targeted to axons, indicative of an ancient role for cannabinoid receptors as axonal regulators of neuronal signalling. Although CB₁/CB₂-type receptors are unique to chordates, enzymes involved in biosynthesis/inactivation of endocannabinoids occur throughout the animal kingdom. Accordingly, non-CB₁/CB₂-mediated mechanisms of endocannabinoid signalling have been postulated. For example, there is evidence that 2-AG mediates retrograde signalling at synapses in the nervous system of the leech Hirudo medicinalis by activating presynaptic transient receptor potential vanilloid-type ion channels. Thus, postsynaptic synthesis of 2-AG or anandamide may be a phylogenetically widespread phenomenon, and a variety of proteins may have evolved as presynaptic (or postsynaptic) receptors for endocannabinoids.     

Ionotropic glutamate receptors

In the brain, most fast excitatory synaptic transmission is mediated through L-glutamate acting on postsynaptic ionotropic glutamate receptors. These receptors are of two kinds—the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)/kainate (non-NMDA) and theN-methyl-D-aspartate (NMDA) receptors, which are thought to be colocalized onto the same postsynaptic elements. This excitatory transmission can be modulated both upward and downward, long-term potentiation (LTP) and long-term depression (LTD), respectively. Whether the expression of LTP/LTD is pre-or postsynaptically located (or both) remains an enigma. This article will focus on what postsynaptic modifications of the ionotropic glutamate receptors may possibly underly long-term potentiation/depression. It will discuss the character of LTP/LTD with respect to the temporal characteristics and to the type of changes that appears in the non-NMDA and NMDA receptor-mediated synaptic currents, and what constraints these findings put on the possible expression mechanism(s) for LTP/LTD. It will be submitted that if a modification of the glutamate receptors does underly LTP/LTD, an increase/decrease in the number of functional receptors is the most plausible alternative. This change in receptor number will have to include a coordinated change of both the non-NMDA and the NMDA receptors.