Depolarization-induced Suppression Of Excitation Research Articles

Cannabinoids regulate an insula circuit controlling water intake

The insular cortex, or insula, is a large brain region involved in the detection of thirst and the regulation of water intake. However, our understanding of the topographical, circuit, and molecular mechanisms for controlling water intake within the insula remains parcellated. We found that type-1 cannabinoid (CB1) receptors in the insular cortex cells participate in the regulation of water intake and deconstructed the circuit mechanisms of this control. Topographically, we revealed that the activity of excitatory neurons in both the anterior insula (aIC) and posterior insula (pIC) increases in response to water intake, yet only the specific removal of CB1 receptors in the pIC decreases water intake. Interestingly, we found that CB1 receptors are highly expressed in insula projections to the basolateral amygdala (BLA), while undetectable in the neighboring central part of the amygdala. Thus, we recorded the neurons of the aIC or pIC targeting the BLA (aIC-BLA and pIC-BLA) and found that they decreased their activity upon water drinking. Additionally, chemogenetic activation of pIC-BLA projection neurons decreased water intake. Finally, we uncovered CB1-dependent short-term synaptic plasticity (depolarization-induced suppression of excitation [DSE]) selectively in pIC-BLA, compared with aIC-BLA synapses. Altogether, our results support a model where CB1 receptor signaling promotes water intake by inhibiting the pIC-BLA pathway, thereby contributing to the fine top-down control of thirst responses.

Mobilization of endocannabinoids by midbrain dopamine neurons is required for the encoding of reward prediction

Brain levels of the endocannabinoid 2-arachidonoylglycerol (2-AG) shape motivated behavior and nucleus accumbens (NAc) dopamine release. However, it is not clear whether mobilization of 2-AG specifically from midbrain dopamine neurons is necessary for dopaminergic responses to external stimuli predicting forthcoming reward. Here, we use a viral-genetic strategy to prevent the expression of the 2-AG-synthesizing enzyme diacylglycerol lipase α (DGLα) from ventral tegmental area (VTA) dopamine cells in adult mice. We find that DGLα deletion from VTA dopamine neurons prevents depolarization-induced suppression of excitation (DSE), a form of 2-AG-mediated synaptic plasticity, in dopamine neurons. DGLα deletion also decreases effortful, cue-driven reward-seeking but has no effect on non-cued or low-effort operant tasks and other behaviors. Moreover, dopamine recording in the NAc reveals that deletion of DGLα impairs the transfer of accumbal dopamine signaling from a reward to its earliest predictors. These results demonstrate that 2-AG mobilization from VTA dopamine neurons is a necessary step for the generation of dopamine-based predictive associations that are required to direct and energize reward-oriented behavior.

Activation of Cannabinoid Receptor 1 in GABAergic Neurons in the Rostral Anterior Insular Cortex Contributes to the Analgesia Following Common Peroneal Nerve Ligation

The rostral agranular insular cortex (RAIC) has been associated with pain modulation. Although the endogenous cannabinoid system (eCB) has been shown to regulate chronic pain, the roles of eCBs in the RAIC remain elusive under the neuropathic pain state. Neuropathic pain was induced in C57BL/6 mice by common peroneal nerve (CPN) ligation. The roles of the eCB were tested in the RAIC of ligated CPN C57BL/6J mice, glutamatergic, or GABAergic neuron cannabinoid receptor 1 (CB1R) knockdown mice with the whole-cell patch-clamp and pain behavioral methods. The E/I ratio (amplitude ratio between mEPSCs and mIPSCs) was significantly increased in layer V pyramidal neurons of the RAIC in CPN-ligated mice. Depolarization-induced suppression of inhibition but not depolarization-induced suppression of excitation in RAIC layer V pyramidal neurons were significantly increased in CPN-ligated mice. The analgesic effect of ACEA (a CB1R agonist) was alleviated along with bilateral dorsolateral funiculus lesions, with the administration of AM251 (a CB1R antagonist), and in CB1R knockdown mice in GABAergic neurons, but not glutamatergic neurons of the RAIC. Our results suggest that CB1R activation reinforces the function of the descending pain inhibitory pathway via reducing the inhibition of glutamatergic layer V neurons by GABAergic neurons in the RAIC to induce an analgesic effect in neuropathic pain.

Differential Enantiomer-Specific Signaling of Cannabidiol at CB1 Receptors

The two main constituents of cannabis are Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD). While Δ9-THC pharmacology has been studied extensively, CBD-long considered inactive-is now the subject of vigorous research related to epilepsy, pain, and inflammation and is popularly embraced as a virtual cure-all. However, our understanding of CBD pharmacology remains limited, although CBD inhibits cannabinoid CB1 receptor signaling, likely as a negative allosteric modulator. Cannabis synthesizes (-)-CBD, but CBD can also exist as an enantiomer, (+)-CBD. We enantioselectively synthesized both CBD enantiomers using established conditions and describe here a new, practical, and reliable, NMR-based method for confirming the enantiomeric purity of two CBD enantiomers. We also investigated the pharmacology of (+)-CBD in autaptic hippocampal neurons, a well-characterized neuronal model of endogenous cannabinoid signaling, and in CHO-K1 cells. We report the inhibition constant for displacing CP55,940 at CB1 by (+)-CBD, is 5-fold lower than (-)-CBD. We find that (+)-CBD is ∼10 times more potent at inhibiting depolarization-induced suppression of excitation (DSE), a form of endogenous cannabinoid-mediated retrograde synaptic plasticity. (+)-CBD also inhibits CB1 suppression of cAMP accumulation but with less potency, indicating that the signaling profiles of the enantiomers differ in a pathway-specific manner. In addition, we report that (+)-CBD stereoselectively and potently activates the sphingosine-1 phosphate (S1P) receptors, S1P1 and S1P3 These results provide an attractive method for synthesizing and distinguishing enantiomers of CBD and related phytocannabinoids and provide further evidence that these enantiomers have their own unique and interesting signaling properties. SIGNIFICANCE STATEMENT: Cannabidiol (CBD) is the subject of considerable scientific and popular interest, but we know little of the enantiomers of CBD. We find that the enantiomer (+)-CBD is substantially more potent inhibitor of cannabinoid CB1 receptors and that it activates sphingosine-1-phosphate receptors in an enantiomer-specific manner; we have additionally developed an improved method for the synthesis of enantiomers of CBD and related compounds.

Cell type specific cannabinoid CB1 receptor distribution across the human and non-human primate cortex

Alterations in cannabinoid CB1 receptor (CB1R) are implicated in various psychiatric disorders. CB1R participates in both depolarization induced suppression of inhibition (DSI) and depolarization induced suppression of excitation (DSE), suggesting its involvement in regulating excitatory and inhibitory (E/I) balance. Prior studies examining neuronal cell type specific CB1R distribution have been conducted near exclusively within rodents. Identification of these distribution patterns within the human and non-human primate cortex is essential to increase our insight into its function. Using co-labeling immunohistochemistry and fluorescent microscopy, we examined CB1R protein levels within excitatory and inhibitory boutons of male human and non-human primate prefrontal cortex and auditory cortices, regions involved in the behavioral effects of exogenous cannabinoid exposures. We found that CB1R was present in both bouton populations within all brain regions examined in both species. Significantly higher CB1R levels were found within inhibitory than within excitatory boutons across all regions in both species, although the cell type by brain region interactions differed between the two species. Our results support the importance of conducting more in-depth CB1R examinations to understand how cell type and brain region dependent differences contribute to regional E/I balance regulation, and how aberrations in CB1R distribution may contribute to pathology.

26.3 Cell-Type–Specific Type 1 Cannabinoid Receptor Distribution Across the Human and Nonhuman Primate Cortex

Alterations in the type-1 cannabinoid (CB1) receptor are implicated in various mental health disorders. CB1 participates in both depolarization-induced suppression of inhibition (DSI) and depolarization-induced suppression of excitation (DSE), suggesting CB1's involvement in both inhibitory and excitatory circuits. However, previous studies describing the distribution of CB1 in human and nonhuman primate cortex utilized antibodies that preferentially target CB1 at inhibitory boutons. Given CB1’s role in both DSI and DSE, understanding cell type–specific CB1 distribution may increase our insight into its regulation of excitatory-inhibitory balance. Here, we investigate CB1 distribution in both inhibitory and excitatory boutons using quantitative fluorescent microscopy. Coronal sections containing dorsolateral prefrontal cortex (DLPFC) and superior temporal gyrus (STG) from adult male macaques and humans are labeled with antibodies for CB1 (validated to target both excitatory and inhibitory boutons), the vesicular glutamate transporter 1 (VGLUT1; labeling intracortical excitatory boutons), and the vesicular GABA transporter (VGAT; labeling intracortical inhibitory boutons). Samples are imaged to visualize protein expression patterns within cortical regions and layers (10 imaged sites per layer for each brain region of each subject). Fluorescent intensities of CB1 label within boutons of each type are compared using multifactorial ANOVA. CB1 co-expression with both VGLUT1 and VGAT are visualized in axons and boutons in all brain regions of both nonhuman primate and human samples. CB1 fluorescent intensity is lower in excitatory boutons compared to inhibitory boutons across all areas (p < 0.01) and exhibits distinct patterns within each brain region and cortical layer. Previous studies describing the distribution of CB1 in human and nonhuman primate cortex utilized antibodies preferentially targeting CB1 at inhibitory boutons. Using a CB1 antibody that targets both excitatory and inhibitory boutons, we demonstrate distinct cell type–specific regional and laminar distributions of CB1 in the human and nonhuman primate cortex. This may increase our understanding of how CB1 differentially regulates excitatory-inhibitory balance within these areas, and whether cell type–specific CB1 disturbances contribute to the development of psychopathology.

Spatial synaptic modulation through IP3 diffusion triggered by ECB: a computational study with an astrocyte-neurons model.

Recently, functional interactions between neurons and astrocytes have been steadily clarified. In particular, the effects of presynaptic depolarization-induced suppression of excitation (DSE) through endocannabinoid (ECB) and endocannabinoids-mediated synaptic potentiation (eSP) by an astrocyte have been used as an evidence of global heterosynaptic modulation. However, the mechanism of occurrence of spatial modulation in a neural network remains unknown. Although the Ca2+ density in astrocytes is strongly related to eSP through ECB, the mechanism of the rise in the ECB receptor in Ca2+ remains unclear. Since Ca2+ is closely related to inositol-1,4,5-trisphosphate (IP3), it is believed that the released IP3 affects Ca2+ in astrocytes that receive ECB. Therefore, this study approximately showed the spatial distribution of DSE or eSP with astrocyte-neuron computational models, assuming that the IP3 caused by ECB is transmitted in an astrocyte. The results showed doughnut-shaped DSE, eSP, and DSE regions from the central ECB released points to the surroundings. They suggested that IP3 diffusion plays an important role in mediating this synaptic modulation.

Depolarization-initiated endogenous cannabinoid release and underlying retrograde neurotransmission in interneurons of amygdala.

The depolarization is also important for the short-term synaptic plasticity, known as depolarization-induced suppression of excitation (DSE). The two major types of neurons and their synapses in the lateral nucleus of amygdala (LA) are prone to plasticity. However, DSE in interneurons has not been reported in amygdala in general and in LA in particular. Therefore, we conducted the patch-clamp experiments with LA interneurons. These neurons were identified by lack of adaptation in firing rate of action potentials. In this study, we show for the first time a transient suppression of neurotransmission at synapses both within the local network and between cortical inputs and interneurons of the LA. The retrograde neurotransmission from GABAergic interneurons were comparable with that of glutamatergic pyramidal cells. That is the axonal terminals of cortical inputs do not posses selectivity toward two neuronal subtypes. However, the DSE of both types of neurons involve an increase in intracellular Ca2+ and the release of endogenous cannabinoids (eCB) and activation of presynaptic CB1 receptors. The magnitude of DSE was significantly higher in interneurons compared with pyramidal cells, though developed with some latency.

Sex differences in the effect of acute fasting on excitatory and inhibitory synapses onto ventral tegmental area dopamine neurons.

Fasting can increase motivation for food and can energize reward-seeking. Ventral tegmental area (VTA) dopamine neurons respond to motivationally relevant information and fasting can influence mesolimbic dopamine concentration. An acute overnight fast differentially alters food approach behaviours and excitatory synaptic transmission onto VTA dopamine neurons of male or female mice. While inhibitory synapses onto VTA dopamine neurons are not altered by fasting in male or female mice, male mice had strengthened excitatory synapses whereas female mice had increased endocannabinoid-mediated short-term plasticity at excitatory synapses. These results help us understand how fasting differentially influences excitatory synaptic transmission onto dopamine neurons and may inform different strategies for fasting-induced food seeking by male and female mice. Dopamine neurons in the ventral tegmental area (VTA) are important for energizing goal-directed behaviour towards food and are sensitive to changes in metabolic states. Fasting increases the incentive motivation for food and the mobilization of energy stores and has sex-dependent effects. However, it is unknown how acute fasting alters excitatory or inhibitory synaptic transmission onto VTA dopamine neurons. An acute 16h overnight fast induced increased food-seeking behaviour that was more predominant in male mice. Fasting increased miniature excitatory postsynaptic current frequency and amplitude in male, but not female, mice. This effect was not due to altered release probability as there was no change in the paired pulse ratio, nor was it due to an altered postsynaptic response as there was no change in the AMPA receptor/NMDA receptor ratio or response to glutamate uncaging. However, this effect was consistent with an increase in the number of release sites. In addition, depolarization-induced suppression of excitation, a measure of short-term endocannabinoid-mediated plasticity, was enhanced in female but not male fasted mice. There were no fasting-induced changes at inhibitory synapses onto dopamine neurons of male or female mice. Taken together, these results demonstrate that fasting influences excitatory synapses differentially in male and female mice, but preserves inhibitory synapses onto dopamine neurons, indicating that the mesolimbic circuits of male and female mice respond differently to acute energy deprivation.

Re-Visiting Type 1 Cannabinoid Receptor Distribution Across the Human and Non-Human Primate Cortex

Alterations in type 1 cannabinoid (CB1) receptor are implicated in psychiatric disorders such as schizophrenia. CB1 participates in both depolarization-induced suppression of inhibition (DSI) and depolarization-induced suppression of excitation (DSE), suggesting its involvement in both inhibitory and excitatory circuits. However, previous studies describing the distribution of CB1 in human and non-human primate cortex utilized antibodies that preferentially targeted CB1 at inhibitory boutons. Given CB1’s role in both DSI and DSE, understanding cell-type specific CB1 distribution may increase our insight into its regulation of excitatory-inhibitory balance. Here, we investigate CB1 distribution in both inhibitory and excitatory boutons using quantitative fluorescent microscopy.

HIV Tat Protein Selectively Impairs CB1 Receptor-Mediated Presynaptic Inhibition at Excitatory But Not Inhibitory Synapses.

Despite the success of antiretroviral therapy in suppressing viral load, nearly half of the 37 million people infected with HIV experience cognitive and motor impairments, collectively classified as HIV-associated neurocognitive disorders (HAND). In the CNS, HIV-infected microglia release neurotoxic agents that act indirectly to elicit excitotoxic synaptic injury. HIV trans-activator of transcription (Tat) protein is one such neurotoxin that is thought to play a major role in the neuropathogenesis of HAND. The endocannabinoid (eCB) system provides on-demand neuroprotection against excitotoxicity, and exogenous cannabinoids attenuate neurotoxicity in animal models of HAND. Whether this neuroprotective system is altered in the presence of HIV is unknown. Here, we examined the effects of Tat on the eCB system in rat primary hippocampal cultures. Using whole-cell patch-clamp electrophysiology, we measured changes in retrograde eCB signaling following exposure to Tat. Treatment with Tat significantly reduced the magnitude of depolarization-induced suppression of excitation (DSE) in a graded manner over the course of 48 h. Interestingly, Tat did not alter this form of short-term synaptic plasticity at inhibitory terminals. The Tat-induced decrease in eCB signaling resulted from impaired CB1 receptor (CB1R)-mediated presynaptic inhibition of glutamate release. This novel loss-of-function was particularly dramatic for low-efficacy agonists such as the eCB 2-arachidonoylglycerol (2-AG) and Δ9-tetrahydrocannabinol (Δ9-THC), the main psychoactive ingredient in marijuana. Our observation that HIV Tat decreases CB1R function in vitro suggests that eCB-mediated neuroprotection may be reduced in vivo; this effect of Tat may contribute to synaptodendritic injury in HAND.

Long-term depression of presynaptic cannabinoid receptor function at parallel fibre synapses.

Inhibition of synaptic responses by activation of presynaptic cannabinoid type-1 (Cb1) receptors is reduced at parallel fibre synapses in the cerebellum following 4Hz stimulation. Activation of adenylyl cyclase is necessary and sufficient for down-regulation of Cb1 receptors induced by 4Hz stimulation. 4Hz stimulation reduces Cb1 receptor function by (i) increasing the rate of endocannabinoid clearance from the synapse and (ii) decreasing expression of Cb1 receptors. Cannabinoid type-1 receptors (Cb1R) are expressed in the presynaptic membrane of many synapses, including parallel fibre-Purkinje cell synapses in the cerebellum, where they are involved in short- and long-term plasticity of synaptic responses. We show that Cb1R expression itself is a plastic property of the synapse regulated by physiological activity patterns. We made patch clamp recordings from Purkinje cells in cerebellar slices and assessed Cb1R activity by measuring depolarization-induced suppression of excitation (DSE). We find that DSE is normally stable at parallel fibre synapses but, following 4Hz stimulation, DSE is persistently reduced and recovers more rapidly. Using a combination of electrophysiology, pharmacology and biochemistry, we show that changes in DSE are a result of the reduced expression of Cb1Rs and increased degradation of endocannabinoids by monoacylglycerol lipase. Long-term changes in presynaptic Cb1R expression may alter other forms of Cb1R-dependent plasticity at parallel fibre synapses, priming or inhibiting the circuit for associative learning.

Impaired anandamide/palmitoylethanolamide signaling in hippocampal glutamatergic neurons alters synaptic plasticity, learning, and emotional responses

Endocannabinoid signaling via anandamide (AEA) is implicated in a variety of neuronal functions and considered a promising therapeutic target for numerous emotion-related disorders. The major AEA degrading enzyme is fatty acid amide hydrolase (FAAH). Genetic deletion and pharmacological inhibition of FAAH reduce anxiety and improve emotional responses and memory in rodents and humans. Complementarily, the mechanisms and impact of decreased AEA signaling remain to be delineated in detail. In the present study, using the Cre/loxP system combined with an adeno-associated virus (AAV)-mediated delivery system, FAAH was selectively overexpressed in hippocampal CA1-CA3 glutamatergic neurons of adult mice. This approach led to specific FAAH overexpression at the postsynaptic site of CA1-CA3 neurons, to increased FAAH enzymatic activity, and, in consequence, to decreased hippocampal levels of AEA and palmitoylethanolamide (PEA), but the levels of the second major endocannabinoid 2-arachidonoyl glycerol (2-AG) and of oleoylethanolamide (OEA) were unchanged. Electrophysiological recordings revealed an enhancement of both excitatory and inhibitory synaptic activity and of long-term potentiation (LTP). In contrast, excitatory and inhibitory long-term depression (LTD) and short-term synaptic plasticity, apparent as depolarization-induced suppression of excitation (DSE) and inhibition (DSI), remained unaltered. These changes in hippocampal synaptic activity were associated with an increase in anxiety-like behavior, and a deficit in object recognition memory and in extinction of aversive memory. This study indicates that AEA is not involved in hippocampal short-term plasticity, or eLTD and iLTD, but modulates glutamatergic transmission most likely via presynaptic sites, and that disturbances in this process impair learning and emotional responses.

Corrigendum: Chronic Inflammatory Pain Impairs mGluR5-Mediated Depolarization-Induced Suppression of Excitation in the Anterior Cingulate Cortex.

The anterior cingulate cortex (ACC) is a critical hub for nociceptive perception and pain-related anxiety. Long-term synaptic plasticity in ACC was found to be important for chronic inflammatory pain and pain-related anxiety. As short-term synaptic plasticity, depolarization-induced suppression of excitation (DSE) is involved in several conditions, such as chronic stress, epilepsy, and autism. However, it is still unknown whether DSE in the ACC is involved in the central sensitization of pain and anxiety. Using a whole-cell patch clamp, calcium imaging, western blot, and behavioral testing, we found that DSE was induced by a 2 s depolarization in postsynaptic pyramidal cells in ACC. DSE was mediated by endocannabinoid signaling and modulated by metabotropic glutamate receptor 5 (mGluR5). DSE was impaired by decreasing expression and dysfunction of mGluR5 in a mouse model of inflammatory pain induced by complete Freund's adjuvant. CDPPB, an mGluR5-positive allosteric modulator, could rescue hypersensitivity and anxiety-like behavior in this pain model. Our results demonstrated that mGluR5-mediated short-term plasticity in ACC may be a critical mechanism for chronic pain, and mGluR5 may potentially serve as a target of pain therapy, including treatments for hyperalgesia and anxiety.

Cannabidiol Inhibits Endocannabinoid Signaling in Autaptic Hippocampal Neurons

Δ9-Tetrahydrocannabinol (THC) and cannabidiol (CBD) are two main cannabinoid constituents of marijuana and hashish. The pharmacology of Δ9-THC has been extensively studied, whereas our understanding of the pharmacology of CBD has remained limited, despite excitement in CBD's potential role in treating certain pediatric epilepsies and its reputation for attenuating some Δ9-THC-induced effects. It was established early on that CBD binds poorly to the orthosteric site of CB1 or CB2 cannabinoid receptors, and its actions were commonly attributed to other noncannabinoid receptor mechanisms. However, recent evidence suggests that CBD does indeed act at cannabinoid CB1 receptors as a negative allosteric modulator (NAM) of CB1 signaling. By altering the orthosteric signaling of a G protein-coupled receptor, allosteric modulators greatly increase the richness of G protein-coupled receptor pharmacology. We have recently surveyed candidate CB1 NAMs in autaptic hippocampal neurons, a well-characterized neuronal model of endogenous cannabinoid signaling, and have now tested CBD in this model. We find that although CBD has no direct effect on excitatory transmission, it does inhibit two forms of endogenous cannabinoid-mediated retrograde synaptic plasticity: depolarization-induced suppression of excitation and metabotropic suppression of excitation, while not affecting signaling via GABA-B receptors. These results are consistent with the recently described NAM activity of CBD and suggest interesting possible mechanisms for CBD's therapeutic actions.

Cannabinoid Receptors Modulate Excitation of an Olfactory Bulb Local Circuit by Cortical Feedback.

Recent studies have provided evidence that corticofugal feedback (CFF) from the olfactory cortex to the olfactory bulb (OB) can significantly impact the state of excitation of output mitral cells (MCs) and tufted cells (TCs) and also modulate neural synchrony. Interpreting these effects however has been complicated by the large number of cell targets of CFF axons in the bulb. Within the granule cell layer (GCL) alone, CFF axons target both GABAergic granule cells (GCs) as well as GABAergic deep short-axon cells (dSACs) that inhibit GCs. Because GCs are a major source of inhibition of MCs/TCs, CFF could be inhibitory to MCs (by exciting GCs) or disinhibitory (by exciting dSACs that inhibit GCs). In this study, we used patch-clamp recordings combined with optogenetic and electrical stimulation methods to investigate the role of presynaptic cannabinoid receptors in regulating CFF pathways, which could alter the weights of inhibition and disinhibition. Recording first from dSACs, we found that the cannabinoid receptor (CB-R) agonist WIN-55212.2 (WIN) reduced excitatory post-synaptic currents (CFF-EPSCs) driven by stimulation of CFF axons. The effects were reversed by the Type 1 CB-R (CB1-R)-specific antagonist SR-141716A. Furthermore, prolonged 5-s depolarizations applied to postsynaptic dSACs effectively reduced CFF-EPSCs in a CB1-R-dependent fashion, providing evidence for depolarization-induced suppression of excitation (DSE) at CFF-to-dSAC synapses. Further analysis indicated that CB1-Rs mediate widespread suppressive effects on synaptic transmission, occurring at CFF synapses onto different dSAC subtypes and CFF synapses onto GCs. Feedforward excitation of dSACs, mediated by MCs/TCs, however, was not impacted by CB1-Rs. In recordings from MCs, performed to examine the net effect of CB1-R activation on GC-to-MC transmission, we found that WIN could both increase and decrease disynaptic inhibition evoked by CFF axon stimulation. The exact effect depended on the size of the inhibitory response, reflecting the local balance of dSAC vs. GC activation. Our results taken together indicate that CB1-Rs can bidirectionally alter the weighting of inhibition and disinhibition of MCs through their effects on CFF pathways.

Enhancement of Endocannabinoid-dependent Depolarization-induced Suppression of Excitation in Glycinergic Neurons by Prolonged Exposure to High Doses of Salicylate

The Dorsal Cochlear Nucleus (DCN) is a region which has been traditionally linked to the genesis of tinnitus, the constant perception of a phantom sound. Sodium salicylate, a COX-2 inhibitor, can induce tinnitus in high doses. Hyperactivity of DCN neurons is observed in several animal models of tinnitus, including salicylate-induced tinnitus. The DCN presents several forms of endocannabinoid (EC)-dependent synaptic plasticity and COX-2 can also participate in the oxidative degradation of ECs. We recently demonstrated that short-term perfusion of sodium salicylate and other inhibitors of both oxidative and hydrolytic EC degradation did not affect depolarization-induced suppression of excitation (DSE), a form of EC-dependent short-term synaptic plasticity. Here, we show that prolonged incubation with high doses of sodium salicylate (1.4 mM) enhances DSE of synapses onto glycinergic DCN interneurons but not those innervating glutamatergic DCN fusiform neurons. This effect was not reproduced with lower doses of salicylate (140 µM) or with ibuprofen, another inhibitor of COX-2. This effect was not observed in the presence of AM251, an antagonist/inverse agonist of cannabinoid CB1 receptors, showing that it was dependent on EC release. Finally we demonstrated that incubation with salicylate potentiated the increase in intracellular calcium during the depolarization. Our results point to an increased inhibition of DCN inhibitory CW neuron during depolarizations, probably by an enhanced EC release during the depolarizations, which is potentially significant for DCN hyperactivity and tinnitus generation.

Synapse-Specific Regulation Revealed at Single Synapses Is Concealed When Recording Multiple Synapses.

Synaptic transmission and its activity-dependent modulation, known as synaptic plasticity, are fundamental processes in nervous system function. Neurons may receive thousands of synaptic contacts, but synaptic regulation may occur only at individual or discrete subsets of synapses, which may have important consequences on the spatial extension of the modulation of synaptic information. Moreover, while several electrophysiological methods are used to assess synaptic transmission at different levels of observation, i.e., through local field potential and individual whole-cell recordings, their experimental limitations to detect synapse-specific modulation is poorly defined. We have investigated how well-known synapse-specific short-term plasticity, where some synapses are regulated and others left unregulated, mediated by astrocytes and endocannabinoid (eCB) signaling can be assessed at different observational levels. Using hippocampal slices, we have combined local field potential and whole-cell recordings of CA3-CA1 synaptic activity evoked by Schaffer collateral stimulation of either multiple or single synapses through bulk or minimal stimulation, respectively, to test the ability to detect short-term synaptic changes induced by eCB signaling. We also developed a mathematical model assuming a bimodal distribution of regulated and unregulated synapses based on realistic experimental data to simulate physiological results and to predict the experimental requirements of the different recording methods to detect discrete changes in subsets of synapses. We show that eCB-induced depolarization-induced suppression of excitation (DSE) and astrocyte-mediated synaptic potentiation can be observed when monitoring single or few synapses, but are statistically concealed when recording the activity of a large number of synapses. These results indicate that the electrophysiological methodology is critical to properly assess synaptic changes occurring in subsets of synapses, and they suggest that relevant synapse-specific regulatory phenomena may be experimentally undetected but may have important implications in the spatial extension of synaptic plasticity phenomena.

Enantiomer-specific positive allosteric modulation of CB1 signaling in autaptic hippocampal neurons

The cannabinoid signaling system is found throughout the CNS and its involvement in several pathological processes makes it an attractive therapeutic target. Because orthosteric CB1 cannabinoid receptor ligands have undesirable adverse effects there has been great interest in the development of allosteric modulators – both negative (NAMs) and positive (PAMs) – of these receptors. NAMs of CB1 appeared first on the scene, followed more recently by PAMs. Because allosteric modulation can vary depending on the orthosteric ligand it is important to study their function in a system that employs endogenous cannabinoids. We have recently surveyed first generation NAMs using cultured autaptic hippocampal neurons. These neurons express depolarization induced suppression of excitation (DSE), a form of synaptic plasticity that is mediated by CB1 and 2-arachidonoyl glycerol (2-AG); they are therefore an excellent neuronal model of endogenous cannabinoid signaling in which to test CB1 modulators.In this study we find that while two related compounds, GAT211 and ZCZ011, each show PAM-like responses in autaptic hippocampal neurons, they also exhibit complex pharmacology. Notably we were able to separate the PAM- and agonist-like responses of GAT211 by examining the enantiomers of this racemic compound: GAT228 and GAT229. We find that GAT229 exhibits PAM-like behavior while GAT228 appears to directly activate the CB1 receptor.Both GAT229 and ZCZ011 represent the first PAMs that we have found to be effective in using this 2-AG utilizing neuronal model system. Because these compounds may exhibit both probe selectivity and biased signaling it will be important to test them with anandamide as well as other signaling pathways.

Chronic Inflammatory Pain Impairs mGluR5-Mediated Depolarization-Induced Suppression of Excitation in the Anterior Cingulate Cortex.

The anterior cingulate cortex (ACC) is a critical hub for nociceptive perception and pain-related anxiety. Long-term synaptic plasticity in ACC was found to be important for chronic inflammatory pain and pain-related anxiety. As short-term synaptic plasticity, depolarization-induced suppression of excitation (DSE) is involved in several conditions, such as chronic stress, epilepsy, and autism. However, it is still unknown whether DSE in the ACC is involved in the central sensitization of pain and anxiety. Using a whole-cell patch clamp, calcium imaging, western blot, and behavioral testing, we found that DSE was induced by a 2 s depolarization in postsynaptic pyramidal cells in ACC. DSE was mediated by endocannabinoid signaling and modulated by metabotropic glutamate receptor 5 (mGluR5). DSE was impaired by decreasing expression and dysfunction of mGluR5 in a mouse model of inflammatory pain induced by complete Freund's adjuvant. CDPPB, an mGluR5-positive allosteric modulator, could rescue hypersensitivity and anxiety-like behavior in this pain model. Our results demonstrated that mGluR5-mediated short-term plasticity in ACC may be a critical mechanism for chronic pain, and mGluR5 may potentially serve as a target of pain therapy, including treatments for hyperalgesia and anxiety.