Endocannabinoid signalling triggered by NMDA receptor-mediated calcium entry into rat hippocampal neurons

Endocannabinoids are released from neurons in activity-dependent manners, act retrogradely on presynaptic CB₁ cannabinoid receptors, and induce short-term or long-term suppression of transmitter release. The endocannabinoid release is triggered by postsynaptic activation of voltage-gated Ca²⁺ channels and/or G_q-coupled receptors such as group I metabotropic glutamate receptors (I-mGluRs) and M₁/M₃ muscarinic receptors. However, the roles of NMDA receptors, which provide another pathway for Ca²⁺ entry into neurons, in endocannabinoid signalling have been poorly understood. In the present study, we investigated the possible contribution of NMDA receptors in endocannabinoid production by recording IPSCs in cultured hippocampal neurons. Under the conditions minimizing the activation of voltage-gated Ca²⁺ channels, local application of NMDA (200 μ m ) transiently suppressed cannabinoid-sensitive IPSCs, but not cannabinoid-insensitive IPSCs. This NMDA-induced suppression was abolished by blocking NMDA receptors, CB₁ receptors and diacylglycerol lipase, but not by inhibiting voltage-gated Ca²⁺ channels. When the postsynaptic neuron was dialysed with 30 m m BAPTA, the NMDA-induced suppression was reduced significantly. A lower dose of NMDA (20 μ m ) exerted little effect when applied alone, but markedly enhanced the cannabinoid-dependent suppression driven by muscarinic receptors or I-mGluRs. These data clearly indicate that the activation of NMDA receptors facilitates the endocannabinoid release either alone or in concert with the G_q-coupled receptors.     

Neurotensin excites periaqueductal gray neurons projecting to the rostral ventromedial medulla

Microinjection of neurotensin into the midbrain periaqueductal gray (PAG) produces a potent and naloxone-insensitive analgesic effect. To test the hypothesis that neurotensin induces the analgesic effect by activating the PAG-rostral ventromedial medulla (RVM) descending antinociceptive pathway, PAG neurons that project to RVM (PAG-RVM) were identified by microinjecting DiI(C18), a retrograde tracing dye, into the rat RVM. Subsequently, fluorescently labeled PAG-RVM projection neurons were acutely dissociated and selected for whole cell patch-clamp recordings. During current-clamp recordings, neurotensin depolarized retrogradely labeled PAG-RVM neurons and evoked action potentials. Voltage-clamp recordings indicated that neurotensin excited PAG-RVM neurons by opening the voltage-insensitive and nonselective cation channels. Both SR 48692, a selective NTR-1 antagonist, and SR 142948A, a nonselective antagonist of NTR-1 and NTR-2, failed to prevent neurotensin from exciting PAG-RVM neurons. Neurotensin failed to evoke cationic currents after internally perfusing PAG-RVM projection neurons with GDP-beta-S or anti-G(alpha q/11) antiserum. Cellular Ca(2+) fluorescence measurement using fura-2 indicated that neurotensin rapidly induced Ca(2+) release from intracellular stores of PAG-RVM neurons. Neurotensin-evoked cationic currents were blocked by heparin, an IP(3) receptor antagonist, and 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), a fast chelator of Ca(2+). These results suggest that by activating a novel subtype of neurotensin receptors, neurotensin depolarizes and excites PAG-RVM projection neurons through enhancing Ca(2+)-dependent nonselective cationic conductance. The coupling mechanism via G(alpha q/11) proteins is likely to involve the production of IP(3), and subsequent IP(3)-evoked Ca(2+) release leads to the opening of nonselective cation channels.     

Cannabinoid-induced presynaptic inhibition at the primary afferent trigeminal synapse of juvenile rat brainstem slices

Systemic or intraventricular administration of cannabinoids causes analgesic effects, but relatively little is known for their cellular mechanism. Using brainstem slices with the mandibular nerve attached, we examined the effect of cannabinoids on glutamatergic transmission in superficial trigeminal caudal nucleus of juvenile rats. The exogenous cannabinoid receptor agonist WIN 55,212-2 (WIN), as well as the endogenous agonist anandamide, hyperpolarized trigeminal caudal neurones and depressed the amplitude of excitatory postsynaptic potentials (EPSPs) or currents (EPSCs) monosynaptically evoked by stimulating mandibular nerves in a concentration-dependent manner. The inhibitory action of WIN was blocked or fully reversed by the CB₁ receptor antagonist SR 141716A. WIN had no effect on the amplitude of miniature excitatory postsynaptic currents (mEPSCs) recorded in the presence of tetrodotoxin or cadmium. The inhibitory effect of WIN on EPSCs was greater for those with longer synaptic latency, suggesting that cannabinoids have a stronger effect on C-fibre EPSPs than on Aδ-fibre EPSPs. Ba²⁺ (100 μ m ) blocked the hyperpolarizing effect of cannabinoids, but did not affect their inhibitory effect on EPSPs. The N-type Ca²⁺ channel blocker ω-conotoxin GVIA (ω-CgTX) occluded the WIN-mediated presynaptic inhibition, whereas the P/Q-type Ca²⁺ channel blocker ω-agatoxin TK (ω-Aga) had no effect. These results suggest that cannabinoids preferentially activate CB₁ receptors at the nerve terminal of small-diameter primary afferent fibres. Upon activation, CB₁ receptors may selectively inhibit presynaptic N-type Ca²⁺ channels and exocytotic release machinery, thereby attenuating the transmitter release at the trigeminal nociceptive synapses.     

Neurotensin enhances GABAergic activity in rat hippocampus CA1 region by modulating L-type calcium channels

Neurotensin (NT) is a tridecapeptide that interacts with three NT receptors; NTS1, NTS2, and NTS3. Although NT has been reported to modulate GABAergic activity in the brain, the underlying cellular and molecular mechanisms of NT are elusive. Here, we examined the effects of NT on GABAergic transmission and the involved cellular and signaling mechanisms of NT in the hippocampus. Application of NT dose-dependently increased the frequency of spontaneous inhibitory postsynaptic currents (sIPSCs) recorded from CA1 pyramidal neurons with no effects on the amplitude of sIPSCs. NT did not change either the frequency or the amplitude of miniature (m)IPSCs recorded in the presence of tetrodotoxin. Triple immunofluorescent staining of recorded interneurons demonstrated the expression of NTS1 on GABAergic interneurons. NT increased the action potential firing rate but decreased the afterhyperpolarization (AHP) amplitude in identified CA1 interneurons. Application of L-type calcium channel blockers (nimodipine and nifedipine) abolished NT-induced increases in action potential firing rate and sIPSC frequency and reduction in AHP amplitude, suggesting that the effects of NT are mediated by interaction with L-type Ca(2+) channels. NT-induced increase in sIPSC frequency was blocked by application of the specific NTS1 antagonist SR48692, the phospholipase C (PLC) inhibitor U73122, the IP(3) receptor antagonist 2-APB, and the protein kinase C inhibitor GF109203X, suggesting that NT increases gamma-aminobutyric acid release via a PLC pathway. Our results provide a cellular mechanism by which NT controls GABAergic neuronal activity in hippocampus.     

Opioid-like actions of neuropeptide Y in rat substantia gelatinosa: Y1 suppression of inhibition and Y2 suppression of excitation

Neuropathic pain that results from injury to the peripheral or CNS responds poorly to opioid analgesics. Y1 and Y2 receptors for neuropeptide Y (NPY) may, however, serve as targets for analgesics that retain their effectiveness in neuropathic pain states. In substantia gelatinosa neurons in spinal cord slices from adult rats, we find that NPY acts via presynaptic Y2 receptors to attenuate excitatory postsynaptic currents (EPSCs) and predominantly on presynaptic Y1 receptors to attenuate glycinergic and GABAergic inhibitory postsynaptic currents (IPSCs). Because NPY attenuates the frequency of TTX-resistant miniature EPSCs and IPSCs, perturbation of the neurotransmitter release process contributes to its actions at both excitatory and inhibitory synapses. These effects, which are reminiscent of those produced by analgesic opioids, provide a cellular basis for previously documented spinal analgesic actions mediated via Y1 and Y2 receptors in neuropathic pain paradigms. They also underline the importance of suppression of inhibition in spinal analgesic mechanisms.     

Dual Ca2+ modulation of glycinergic synaptic currents in rodent hypoglossal motoneurones

Glycinergic synapses are implicated in the coordination of reflex responses, sensory signal processing and pain sensation. Their activity is pre- and postsynaptically regulated, although mechanisms are poorly understood. Using patch-clamp recording and Ca²⁺ imaging in hypoglossal motoneurones from rat and mouse brainstem slices, we address here the role of cytoplasmic Ca²⁺ (Ca_i) in glycinergic synapse modulation. Ca²⁺ influx through voltage-gated or NMDA receptor channels caused powerful transient inhibition of glycinergic IPSCs. This effect was accompanied by an increase in both the failure rate and paired-pulse ratio, as well as a decrease in the frequency of mIPSCs, suggesting a presynaptic mechanism of depression. Inhibition was reduced by the cannabinoid receptor antagonist SR141716A and occluded by the agonist WIN55,212-2, indicating involvement of endocannabinoid retrograde signalling. Conversely, in the presence of SR141716A, glycinergic IPSCs were potentiated postsynaptically by glutamate or NMDA, displaying a Ca²⁺-dependent increase in amplitude and decay prolongation. Both presynaptic inhibition and postsynaptic potentiation were completely prevented by strong Ca_i buffering (20 m m BAPTA). Our findings demonstrate two independent mechanisms by which Ca²⁺ modulates glycinergic synaptic transmission: (i) presynaptic inhibition of glycine release and (ii) postsynaptic potentiation of GlyR-mediated responses. This dual Ca²⁺-induced regulation might be important for feedback control of neurotransmission in a variety of glycinergic networks in mammalian nervous systems.     

Synaptic GABAergic and glutamatergic mechanisms underlying alcohol sensitivity in mouse hippocampal neurons

This study was designed to examine the neuronal mechanisms of ethanol sensitivity by utilizing inbred short sleep (ISS) and inbred long sleep (ILS) mouse strains that display large differences in sensitivity to the behavioural effects of ethanol. Comparisons of whole-cell electrophysiological recordings from CA1 pyramidal neurons in hippocampal slices of ISS and ILS mice indicate that ethanol enhances GABA_A receptor-mediated inhibitory postsynaptic currents (GABA_A IPSCs) and reduces NMDA receptor-mediated excitatory postsynaptic currents (NMDA EPSCs) in a concentration- and strain-dependent manner. In ILS neurons, these receptor systems are significantly more sensitive to ethanol than those in ISS neurons. To further examine the underlying mechanisms of differential ethanol sensitivities in these mice, GABA_B activity and presynaptic and postsynaptic actions of ethanol were investigated. Inhibition of GABA_B receptor function enhances ethanol-mediated potentiation of distal GABA_A IPSCs in ILS but not ISS mice, and this blockade of GABA_B receptor function has no effect on the action of ethanol on NMDA EPSCs in either mouse strain. Thus, subregional differences in GABA_B activity may contribute to the differential ethanol sensitivity of ISS and ILS mice. Moreover, analysis of the effects of ethanol on paired-pulse stimulation, spontaneous IPSC events, and brief local GABA or glutamate application suggest that postsynaptic rather than presynaptic mechanisms underlie the differential ethanol sensitivity of these mice. Furthermore, these results provide essential information to focus better on appropriate target sites for more effective drug development for the treatment of alcohol abuse.     

GABAB Receptor- and Metabotropic Glutamate Receptor-Dependent Cooperative Long-Term Potentiation of Rat Hippocampal GABAA Synaptic Transmission

Repetitive stimulation of Schaffer collaterals induces activity-dependent changes in the strength of polysynaptic inhibitory postsynaptic potentials (IPSPs) in hippocampal CA1 pyramidal neurons that are dependent on stimulation parameters. In the present study, we investigated the effects of two stimulation patterns, theta-burst stimulation (TBS) and 100 Hz tetani, on pharmacologically isolated monosynaptic GABAergic responses in adult CA1 pyramidal cells. Tetanization with 100 Hz trains transiently depressed both early and late IPSPs, whereas TBS induced long-term potentiation (LTP) of early IPSPs that lasted at least 30 min. Mechanisms mediating this TBS-induced potentiation were examined using whole-cell recordings. The paired-pulse ratio of monosynaptic inhibitory postsynaptic currents (IPSCs) was not affected during LTP, suggesting that presynaptic changes in GABA release are not involved in the potentiation. Bath application of the GABA_B receptor antagonist CGP55845 or the group I/II metabotropic glutamate receptor antagonist E4-CPG inhibited IPSC potentiation. Preventing postsynaptic G-protein activation or Ca²⁺ rise by postsynaptic injection of GDP-β-S or BAPTA, respectively, abolished LTP, indicating a G-protein- and Ca²⁺-dependent induction in this LTP. Finally during paired-recordings, activation of individual interneurons by intracellular TBS elicited solely short-term increases in average unitary IPSCs in pyramidal cells. These results indicate that a stimulation paradigm mimicking the endogenous theta rhythm activates cooperative postsynaptic mechanisms dependent on GABA_BR, mGluR, G-proteins and intracellular Ca²⁺, which lead to a sustained potentiation of GABA_A synaptic transmission in pyramidal cells. GABAergic synapses may therefore contribute to functional synaptic plasticity in adult hippocampus.     

The effects of diphenhydramine and SR142948A on periaqueductal gray neurons and on the interactions between the medial preoptic nucleus and the periaqueductal gray

The midbrain periaqueductal gray contains both neurotensin type-1 and type-2 receptors. Behavioral studies have shown that the analgesic effect of neurotensin is mediated through its interaction with the type-2 receptors. These receptors specifically bind the type-1 histamine antagonist, levocabastine. Recently, it has been shown that another histamine-1 antagonist, diphenhydramine, blocks the analgesic effect of neurotensin. In addition, it has been shown that a non-peptide neurotensin antagonist, SR142948A, binds to both types of neurotensin receptors and blocks the analgesic effect of exogenously applied neurotensin. Major afferents to the periaqueductal gray arise from the medial preoptic nucleus of the hypothalamus. This region contains neurotensinergic neurons, and the expression of neurotensin mRNA in this region increases following cold-water swim stress that leads to opioid-independent analgesia. The goal of this study was to determine whether the responses of periaqueductal gray neurons to stimulation of the medial preoptic nucleus are modified by local injection of diphenhydramine and SR142948A. Because the cellular basis of the effects of diphenhydramine on periaqueductal gray neurons had not been reported, we also examined the effects of diphenhydramine on the baseline-firing rate and synaptic transmission using in vivo and in vitro methods. The results of the in vitro studies indicate that diphenhydramine concentrations above 500 nM significantly reduce the baseline firing of the periaqueductal gray neurons without a significant effect on the frequency of postsynaptic potentials. At concentrations below 100 nM, diphenhydramine has little effect on the baseline-firing rate but partially blocks the response to neurotensin. The results of the in vivo studies showed similar effects of diphenhydramine. At high concentrations it inhibited periaqueductal gray neurons, but at low concentrations it had no effect on the baseline-firing rate and it blocked the response to neurotensin and to medial preoptic nucleus stimulation. Unlike diphenhydramine, SR142948A had virtually no effect on the baseline-firing rate but blocked the response to neurotensin and to stimulation of the medial preoptic nucleus. It is concluded that: (1) SR142948A, at a dose that completely blocks the effect of exogenously applied neurotensin on periaqueductal gray neurons, has little effect on their baseline-firing rates. (2) Because of its effect on the baseline-firing rate, only low doses of diphenhydramine can be used as an antagonist of the neurotensin analgesic effect. (3) Responses of periaqueductal gray neurons to medial preoptic nucleus stimulation is, in part, mediated by a neurotensinergic network within the periaqueductal gray.     

Differential ontogeny of GABABreceptor-mediated pre- and postsynaptic modulation of GABA and glycine transmission in respiratory rhythm-generating network in mouse

Rhythm generation in mature respiratory networks is influenced strongly by synaptic inhibition. In early neonates, GABA_A-receptor- and glycine-receptor-mediated inhibition is not present, thus the question arises as to whether GABA_B-receptor-mediated inhibition plays an important role. Using brainstem slices of neonatal mice (postnatal day, P0-P15), we analysed the role of GABA_B-mediated modulation of GABA and glycine synaptic transmission in the respiratory network. Blockade of GABA uptake by nipecotic acid (0.25–2 m m ) reduced the respiratory frequency. This reduction was prevented by the selective GABA_B receptor antagonist CGP55845A (CGP) alone at P0-P3, but by bicuculline as well as CGP at P7-P15. Blockade of GABA_B receptors by CGP increased the respiratory frequency at P0-P3, whereas it caused a reduction of frequency in older animals. The effect of CGP on respiratory frequency was diminished in the presence of bicuculline and strychnine in older but not in younger animals. The relative contribution of GABA_B-receptor-mediated pre- and postsynaptic modulation was examined by analysing the effect of GABA_B receptors on spontaneous and miniature IPSCs. In younger animals (P0-P3), the GABA_B receptor agonist baclofen had no detectable effect on IPSC frequency, but caused a significant decrease in the amplitude. In older animals (P7-P15), baclofen decreased both the frequency and amplitude of spontaneous and miniature IPSCs. These results demonstrate that GABA_B-receptor-mediated postsynaptic modulation plays an important role in the respiratory network from P0 on. GABA_B-receptor-mediated presynaptic modulation develops with a longer postnatal latency, and becomes predominant within the first postnatal week.     

Inspiratory bursts in the preBötzinger complex depend on a calcium-activated non-specific cation current linked to glutamate receptors in neonatal mice

Inspiratory neurons of the preBötzinger complex (preBötC) form local excitatory networks and display 10–30 mV transient depolarizations, dubbed inspiratory drive potentials , with superimposed spiking. AMPA receptors are critical for rhythmogenesis under normal conditions in vitro but whether other postsynaptic mechanisms contribute to drive potential generation remains unknown. We examined synaptic and intrinsic membrane properties that generate inspiratory drive potentials in preBötC neurons using neonatal mouse medullary slice preparations that generate respiratory rhythm. We found that NMDA receptors, group I metabotropic glutamate receptors (mGluRs), but not group II mGluRs, contributed to inspiratory drive potentials. Subtype 1 of the group I mGluR family (mGluR1) probably regulates a K⁺ channel, whereas mGluR5 operates via an inositol 1,4,5-trisphosphate (IP₃) receptor-dependent mechanism to augment drive potential generation. We tested for and verified the presence of a Ca²⁺-activated non-specific cation current ( I _CAN) in preBötC neurons. We also found that high concentrations of intracellular BAPTA, a high-affinity Ca²⁺ chelator, and the I _CAN antagonist flufenamic acid (FFA) decreased the magnitude of drive potentials. We conclude that I _CAN underlies robust inspiratory drive potentials in preBötC neurons, and is only fully evoked by ionotropic and metabotropic glutamatergic synaptic inputs, i.e. by network activity.     

In vitro and in vivo analysis of the Effects of corticotropin releasing factor on rat dorsal vagal complex

In vivo and in vitro electrophysiological experiments were performed on the rat dorsal vagal complex (DVC, i.e. nucleus of the tractus solitarius, NTS, and dorsal motor nucleus of the vagus, DMV) to examine the effects of corticotropin releasing hormone (CRF) on the central components of the vago-vagal reflex control of gastric function. When applied to gastrointestinal projecting DMV neurones, CRF (10-300 n m ) induced a concentration-dependent membrane depolarization, an increase in action potential firing rate and decrease in amplitude of the action potential afterhyperpolarization ( P < 0.05). Pretreatment with the non-selective CRF antagonist, astressin (0.5-1 μM) or the selective CRF₂ receptor antagonist, astressin 2B (500 n m ) attenuated the CRF-induced increase in firing rate but did not alter basal discharge rate. CRF (30-300 n m ) increased the amplitude of excitatory postsynaptic currents (EPSCs) evoked by stimulation of the NTS ( P < 0.05). An alteration in the paired pulse ratio indicated the EPSC's increase occurred due to actions at presynaptic sites. In the in vivo anaesthetized rat preparation, bilateral microinjections (20 fmol in 20 nl for each site) of CRF in the DVC decreased gastric motility in rats pretreated with the muscarinic agonist, bethanecol ( P < 0.05). The effects of CRF were abolished by systemic administration of the NOS inhibitor, L -NAME, or by bilateral vagotomy. We concluded that CRF had both a direct and an indirect excitatory effect on DMV neurones via activation of CRF₂ receptors and the decrease in gastric motility observed following microinjection of CRF in the DVC is due to the activation of an inhibitory non-adrenergic non-cholinergic input to the gastrointestinal tract.     

Pacemaker phase shift in the absence of neural activity in guinea-pig stomach: a microelectrode array study

The nucleus tractus solitarii (NTS) is the first site of integration for primary baroreceptor afferents, which release glutamate to excite second-order neurones through ionotropic receptors. In vitro studies indicate that glutamate may also activate metabotropic receptors (mGluRs) to modulate the excitability of NTS neurones at pre- and postsynaptic loci. We examined the functional role of metabotropic glutamate receptors (mGluRs) in modulating the baroreceptor reflex in the rat NTS. Using the working heart–brainstem preparation, the baroreflex was activated using brief pressor stimuli and the consequent cardiac (heart rate change) and non-cardiac sympathetic (T8–10 chain) baroreflex gains were obtained. Microinjections of glutamate antagonists were made bilaterally into the NTS at the site of termination of baroreceptor afferents. NTS microinjection of kynurenate (ionotropic antagonist) inhibited both the cardiac and sympathetic baroreflex gains (16 ± 5% and 59 ± 11% of control, respectively). The non-selective mGluR antagonist MCPG produced a dose-dependent inhibition of the cardiac gain (30 ± 3% of control) but not the sympathetic gain. Selective inhibitions of the cardiac gain were also seen with LY341495 and EGLU suggesting the response was mediated by group II mGluRs. This effect on cardiac gain involves attenuation of the parasympathetic baroreflex as it persists in the presence of atenolol. Prior NTS microinjection of bicuculline (GABA_A antagonist) prevented the mGluR-mediated attenuation of the cardiac gain. These results are consistent with the reported presynaptic inhibition of GABAergic transmission by group II mGluRs in the NTS and constitute a plausible mechanism allowing selective feed-forward disinhibition to increase the gain of the cardiac limb of the baroreflex without changing the sympathoinhibitory component.     

Group II metabotropic glutamate receptor modulation of excitatory transmission in rat subthalamic nucleus

Patch pipettes were used to record currents in whole-cell configuration to study the effects of group II metabotropic glutamate receptor (mGluR) stimulation on synaptic transmission in slices of rat subthalamic nucleus. Evoked glutamatergic excitatory postsynaptic currents (EPSCs) were reversibly reduced by the selective group II mGluR agonist (2' S ,2' R ,3' R )-2-(2',3'-dicarboxycyclopropyl)glycine (DCG IV) in a concentration-dependent manner, with an IC₅₀ of 0.19 ± 0.05 µ m . DCG IV (1 µ m ) had no effect on inhibitory postsynaptic currents mediated by GABA. DCG IV-induced inhibition of EPSCs was reversed by the selective group II mGluR antagonist LY 341495 (100 n m ) and mimicked by another selective group II agonist (2 S ,1' S ,2' S )-2-(carboxycyclopropyl)glycine ( l -CCG-I). Inhibition of EPSC amplitude by DCG IV and l -CCG-I was associated with an increase in the paired-pulse ratio of EPSCs. The protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (2 µ m ) reduced the inhibitory effect of DCG IV on EPSCs. However, the response to DCG IV was not affected by the protein kinase A (PKA) activator forskolin (20 µ m ), by the adenylyl cyclase inhibitor MDL 12230A (20 µ m ), or by the phosphodiesterase inhibitor Ro 20–1724 (50 µ m ). DCG IV-induced inhibition of EPSCs was reduced by the non-selective protein kinase inhibitors H-7 (100 µ m ), H-8 (50 µ m ) and HA-1004 (100 µ m ). These results suggest that group II mGluR stimulation acts presynaptically to inhibit glutamate release by a PKC-dependent mechanism in the subthalamic nucleus.     

PGE2 glycerol ester, a COX-2 oxidative metabolite of 2-arachidonoyl glycerol, modulates inhibitory synaptic transmission in mouse hippocampal neurons

The oxygenation of endogenous cannabinoids (eCBs) 2-arachidonoyl glycerol (2-AG) and arachidonoyl ethanolamide by cyclooxygenase-2 (COX-2) produces novel types of prostanoids: prostaglandin glycerol esters (PG-Gs) and prostaglandin ethanolamides (PG-EAs). However, the physiological function of COX-2 oxidative metabolites of eCBs is still unclear. Here we demonstrate that PGE₂-G, a COX-2 oxidative metabolite of 2-AG, induced a concentration-dependent increase in the frequency of miniature inhibitory postsynaptic currents (mIPSCs) in primary cultured hippocampal neurons, an effect opposite to that of 2-AG. This increase was not inhibited by SR141716, a CB₁ receptor antagonist, but was attenuated by an IP₃ or MAPK inhibitor. In addition, we also examined the effects of other prostanoids derived from COX-2 oxygenation of eCBs on mIPSCs. PGD₂-G, PGF_2α-G and PGD₂-EA, but not PGE₂-EA or PGF_2α-EA, also increased the frequency of mIPSCs. The eCB-derived prostanoid-induced responses appeared to be different from those of corresponding arachidonic acid-derived prostanoids, implying that these effects are not mediated via known prostanoid receptors. We further discovered that the inhibition of COX-2 activity reduced inhibitory synaptic activity and augmented depolarization-induced suppression of inhibition (DSI), whereas the enhancement of COX-2 augmented the synaptic transmission and abolished DSI. Our results, which show that COX-2 oxidative metabolites of eCBs exert opposite effects to their parent molecules on inhibitory synaptic transmission, suggest that alterations in COX-2 activity will have significant impact on endocannabinoid signalling in hippocampal synaptic activity.     

Dendritically released transmitters cooperate via autocrine and retrograde actions to inhibit afferent excitation in rat brain

Oxytocin is released from supraoptic magnocellular neurones and is thought to act at presynaptic receptors to inhibit transmitter release. We now show that this effect is mediated by endocannabinoids, but that oxytocin nonetheless plays an important role in endocannabinoid signalling. WIN55,212-2, a cannabinoid receptor agonist, mimicked the action of oxytocin and occluded oxytocin-induced presynaptic inhibition. The cannabinoid action is at the presynaptic terminal as shown by alteration in paired pulse ratio, a reduction in miniature EPSC frequency and immunohistochemical localization of CB₁ receptors on presynaptic terminals. AM251, a CB₁ receptor antagonist, blocked both the WIN55,212-2 and the oxytocin-induced presynaptic inhibition of EPSCs. Depolarization of postsynaptic magnocellular neurones (which contain fatty acid amide hydrolase, a cannabinoid catabolic enzyme) caused a transient inhibition of EPSCs that could be blocked by both the AM251 and Manning compound, an oxytocin/vasopressin receptor antagonist. This indicates that somatodendritic peptide release and action on previously identified autoreceptors facilitates the release of endocannabinoids that act as mediators of presynaptic inhibition.     

Involvement of Src tyrosine kinase and mitogen-activated protein kinase in the facilitation of calcium channels in rat nucleus of the tractus solitarius by angiotensin II

It is recognized that brain contains all the components of the renin–angiotensin systems (RAS). The nucleus of the tractus solitarius (NTS) is known to play a major role in the regulation of cardiovascular, respiratory, gustatory, hepatic and swallowing functions. Voltage-dependent Ca²⁺ channels (VDCCs) serve as crucial mediators of membrane excitability and Ca²⁺-dependent functions such as neurotransmitter release, enzyme activity and gene expression. The purpose of this study was to investigate the effects of angiotensin II (Ang II) on VDCC currents ( I _Ca) in the NTS using patch-clamp recording methods. An application of Ang II caused facilitation of L-type I _Ca in a concentration-dependent manner with an EC₅₀ of 167 n m and a Hill coefficient of 1.73. AT₁ receptor antagonist losartan antagonized the Ang II-induced facilitation of I _Ca. Intracellular dialysis of the Gα_i-protein antibody attenuated the Ang II-induced facilitation of I _Ca. Both Src tyrosine kinase inhibitor and mitogen-activated protein kinase (MAPK) inhibitor attenuated the Ang II-induced facilitation of I _Ca. p38 MAPK inhibitor also attenuated the Ang II-induced facilitation of I _Ca. These results indicate that Ang II facilitates L-type VDCCs via Gα_i-proteins involving Src tyrosine kinase and p38 MAPK kinase mediated by AT₁ receptors in NTS.     

Actions of noradrenaline on substantia gelatinosa neurones in the rat spinal cord revealed by in vivo patch recording

To elucidate the mechanisms of antinociception mediated by the descending noradrenergic pathway in the spinal cord, the effects of noradrenaline (NA) on noxious synaptic responses of substantia gelatinosa (SG) neurones, and postsynaptic actions of NA were investigated in rats using an in vivo whole-cell patch-clamp technique. Under urethane anaesthesia, the rat was fixed in a stereotaxic apparatus after the lumbar spinal cord was exposed. In the current-clamp mode, pinch stimuli applied to the ipsilateral hindlimb elicited a barrage of EPSPs, some of which initiated an action potential. Perfusion with NA onto the surface of the spinal cord hyperpolarized the membrane (5.0–9.5 mV) and suppressed the action potentials. In the voltage-clamp mode ( V _H, −70 mV), the application of NA produced an outward current that was blocked by Cs⁺ and GDP-β-S added to the pipette solution and reduced the amplitude of EPSCs evoked by noxious stimuli. Under the blockade of postsynaptic actions of NA, a reduction of the evoked and spontaneous EPSCs of SG neurones was still observed, thus suggesting both pre- and postsynaptic actions of NA. The NA-induced outward currents showed a clear dose dependency (EC₅₀, 20 μ m ), and the reversal potential was −88 mV. The outward current was mimicked by an α₂-adrenoceptor agonist, clonidine, and suppressed by an α₂-adrenoceptor antagonist, yohimbine, but not by α₁- and β-antagonists. These findings suggest that NA acts on presynaptic sites to reduce noxious stimuli-induced EPSCs, and on postsynaptic SG neurones to induce an outward current by G-protein-mediated activation of K⁺ channels through α₂-adrenoceptors, thereby producing an antinociceptive effect.     

Transient receptor potential-like channels mediate metabotropic glutamate receptor EPSCs in rat dopamine neurones

Transient receptor potential (TRP) channels form cationic channels activated by diverse factors including mechanical stimuli, changes in osmolarity, pH and temperature, as well as the exogenous irritant, capsaicin. Metabotropic glutamate receptors have also recently been linked to TRP channel activation in neurones of the substantia nigra, hippocampus and cerebellum, suggesting a novel role for such channels in synaptic communication via endogenous neurotransmitters. We tested this for dopamine neurones in rat brain slices by characterizing the current–voltage relationship and pharmacology of EPSCs mediated by group I metabotropic glutamate receptor subtype 1 (mGluR1). Slow inward currents (273 ± 35 pA peak amplitude, 381 ± 25 ms latency, holding potential ( V _h) =−73 mV) representing evoked mGluR1 EPSCs were isolated in the presence of antagonists of AMPA, NMDA, GABA_A, GABA_B, muscarinic and glycine receptors. CPCCOEt (100 μ m ), an mGluR1 antagonist, blocked the residual EPSC in all recordings. mGluR1-activated EPSCs reversed polarity near −10 mV, consistent with the involvement of a cationic channel. Extracellular application of the non-selective TRP channel blockers SKF 96365, flufenamic acid and ruthenium red caused reversible inhibition of mGluR1-activated EPSCs. These characteristics parallel those of mGluR1 activation with an agonist and indicate the involvement of a TRP-like channel in mGluR1-mediated EPSCs.     

Fast IPSCs in rat thalamic reticular nucleus require the GABAA receptor β1 subunit

Synchrony within the thalamocortical system is regulated in part by intranuclear synaptic inhibition within the reticular nucleus (RTN). Inhibitory postsynaptic currents (IPSCs) in RTN neurons are largely characterized by slow decay kinetics that result in powerful and prolonged suppression of spikes. Here we show that some individual RTN neurons are characterized by highly variable mixtures of fast, slow and mixed IPSCs. Heterogeneity arose largely through differences in the contribution of an initial decay component (τ_D∼10 ms) which was insensitive to loreclezole, suggesting involvement of the GABA_A receptor β₁ subunit. Single-cell RT-PCR revealed the presence of β₁ subunit mRNA only in those neurons whose IPSCs were dominated by a rapid and prominent initial decay phase. These data show that brief, β₁-dependent, loreclezole-insensitive IPSCs are present in a subpopulation of RTN neurons, and suggest that striking differences in IPSC heterogeneity within single neurons can result from of the presence or absence of a single GABA_A receptor subunit.