Endocannabinoids control vesicle release mode at midbrain periaqueductal grey inhibitory synapses.

KEY POINTS: The midbrain periaqueductal grey (PAG) forms part of an endogenous analgesic system which is tightly regulated by the neurotransmitter GABA. The role of endocannabinoids in regulating GABAergic control of this system was examined in rat PAG slices. Under basal conditions GABAergic neurotransmission onto PAG output neurons was multivesicular. Activation of the endocannabinoid system reduced GABAergic inhibition by reducing the probability of release and by shifting release to a univesicular mode. Blockade of endocannabinoid system unmasked a tonic control over the probability and mode of GABA release. These findings provides a mechanistic foundation for the control of the PAG analgesic system by disinhibition. ABSTRACT: The midbrain periaqueductal grey (PAG) has a crucial role in coordinating endogenous analgesic responses to physiological and psychological stressors. Endocannabinoids are thought to mediate a form of stress-induced analgesia within the PAG by relieving GABAergic inhibition of output neurons, a process known as disinhibition. This disinhibition is thought to be achieved by a presynaptic reduction in GABA release probability. We examined whether other mechanisms have a role in endocannabinoid modulation of GABAergic synaptic transmission within the rat PAG. The group I mGluR agonist DHPG ((R,S)-3,5-dihydroxyphenylglycine) inhibited evoked IPSCs and increased their paired pulse ratio in normal external Ca2+ , and when release probability was reduced by lowering Ca2+ . However, the effect of DHPG on the coefficient of variation and kinetics of evoked IPSCs differed between normal and low Ca2+ . Lowering external Ca2+ had a similar effect on evoked IPSCs to that observed for DHPG in normal external Ca2+ . The low affinity GABAA receptor antagonist TPMPA ((1,2,5,6-tetrahydropyridin-4-yl)methylphosphinic acid) inhibited evoked IPSCs to a greater extent in low than in normal Ca2+ . Together these findings indicate that the normal mode of GABA release is multivesicular within the PAG, and that DHPG and lowering external Ca2+ switch this to a univesicular mode. The effects of DHPG were mediated by mGlu5 receptor engagement of the retrograde endocannabinoid system. Blockade of endocannabinoid breakdown produced a similar shift in the mode of release. We conclude that endocannabinoids control both the mode and the probability of GABA release within the PAG.

Substance P drives endocannabinoid-mediated disinhibition in a midbrain descending analgesic pathway.

Substance P is thought to play an essential role in several forms of supraspinally mediated analgesia. The actions of substance P on synaptic transmission within descending analgesic pathways, however, are largely unknown. Here, we used whole-cell recordings from rat midbrain slices to examine the effects of substance P on GABAergic and glutamatergic transmission within the periaqueductal gray (PAG), a key component of a descending analgesic pathway that projects via the rostral ventromedial medulla (RVM) to the spinal cord dorsal horn. We found that substance P reversibly decreased the amplitude and increased the paired-pulse ratio of evoked IPSCs recorded from identified PAG-RVM projection neurons and from unidentified PAG neurons. Substance P had no effect on miniature IPSCs, implying an indirect mode of action. The effects of substance P were abolished by metabotropic glutamate type 5 and cannabinoid CB1 receptor antagonists, but unaltered by NMDA, GABA(B), mu,delta-opioid, adenosine A(1), and 5HT(1A) receptor antagonists. Consistent with a role for endogenous glutamate in this process, substance P increased the frequency of action potential-dependent spontaneous EPSCs. Moreover, the effect of substance P on evoked IPSCs was mimicked and occluded by a glutamate transport inhibitor. Finally, these effects were dependent on postsynaptic G-protein activation and diacylglycerol lipase activity, suggesting the requirement for retrograde signaling by the endocannabinoid 2-arachidonoylglycerol. Thus, substance P may facilitate descending analgesia in part by enhancing glutamate-mediated excitation and endocannabinoid-mediated disinhibition of PAG-RVM projection neurons.

Glutamate spillover modulates GABAergic synaptic transmission in the rat midbrain periaqueductal grey via metabotropic glutamate receptors and endocannabinoid signaling.

Glutamate spillover regulates GABAergic synaptic transmission at several CNS synapses via presynaptic ionotropic and metabotropic glutamate receptors (mGluRs). We have previously demonstrated that activation of group I-III mGluRs inhibits GABAergic transmission in the midbrain periaqueductal gray (PAG), a region involved in organizing behavioral responses to threat, stress, and pain. Here, we examined the role of glutamate spillover in the modulation of GABAergic transmission in the PAG. Using whole-cell recordings from rat PAG slices, we found that evoked IPSCs were reduced by the nonspecific glutamate transport blockers DL-threo-beta-benzyloxyaspartic acid (TBOA) and L-trans-pyrrolidine-2,4-dicarboxylic acid, but not by the glial GLT1-specific blocker dihydrokainate. In contrast, TBOA had no effect on evoked IPSCs when glutamate uptake into the postsynaptic neuron was selectively impaired. TBOA increased the paired-pulse ratio of evoked IPSCs and reduced the rate but not the amplitude of spontaneous miniature IPSCs. The effect of TBOA on evoked IPSCs was abolished by the broad-spectrum mGluR antagonist (2S)-2-amino-2-[(1S,2S)-2-carboxycycloprop-1-yl]-3-(xanth-9-yl) propanoic acid (100 microM), reduced by the mGluR5-specific antagonist 2-methyl-6-(phenylethynyl)pyridine hydrochloride (MPEP) and mimicked by the mGluR1/5 agonist (RS)-3,5-dihydroxyphenylglycine (DHPG). Furthermore, the effects of both TBOA and DHPG were reduced by the cannabinoid CB1 receptor antagonist 1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-1-piperidinyl-1H-pyrazole-3-carboxamide (AM251). Finally, although MPEP and AM251 had no effect on single evoked IPSCs, they increased evoked IPSCs during repetitive stimulation. These results indicate that neuronal glutamate transporters limit mGluR5 activation and endocannabinoid signaling, but may be overwhelmed during conditions of elevated glutamate release. Thus, neuronal glutamate transporters play a key role in regulating endocannabinoid-mediated cross talk between glutamatergic and GABAergic synapses within the PAG.

Postsynaptic actions of substance P on rat periaqueductal grey neurons in vitro.

The postsynaptic actions of substance P on rat midbrain periaqueductal grey (PAG) neurons were examined using whole-cell patch-clamp recordings in brain slices. Substance P produced an inward current in a subpopulation (60%) of PAG neurons. The substance P induced current was concentration dependent (EC50=27 nM) and was reduced by the NK1, NK2 and NK3 antagonists L-732,138 (20 microM), GR 159897 (3 microM) and SB 218795 (3 microM). The selective NK1, NK2 and NK3 agonists [Sar9,Met(O2)11]-Substance P (100 nM), GR 64349 (300-500 nM) and senktide (300 nM) also produced inward currents in subpopulations of neurons. A greater proportion of substance P-sensitive neurons (70%) than substance P-insensitive neurons (31%) responded to the mu/delta opioid agonist met-enkephalin (10 microM). Substance P reduced the outward current produced by met-enkephalin. The reversal potential of the substance P induced current varied from -5 mV to below -140 mV in the absence of met-enkephalin, and was -105 mV in the presence of met-enkephalin. These results indicate that substance P acts via NK1, NK2 and NK3 receptors to excite subpopulations of opioid-sensitive and insensitive PAG neurons by increasing a non-selective cation conductance and by reducing a K+ current. In addition, substance P has anti-opioid actions that are largely mediated by a reduction in the opioid induced K+ current.

Mechanical allodynia following contusion injury of the rat spinal cord is associated with loss of GABAergic inhibition in the dorsal horn.

The present study investigated whether mechanical allodynia following contusive spinal cord injury (SCI) of the thoracic segments 12 and 13 of the rat was associated with a reduction in gamma-aminobutyric acid (GABA)ergic inhibition adjacent to the site of injury. Five to 7 days following SCI, extracellular recordings were obtained from dorsal horn neurones located 1-2 segments caudal to the injury, in non-allodynic and allodynic halothane anaesthetised rats and from comparable neurones in normal rats. To assess spinal GABAergic inhibition in the three groups of animals, spontaneous and evoked cell firing rates were recorded before, during and after microiontophoretic application of the GABA(A) receptor antagonist bicuculline. Administration of bicuculline to normal animals resulted in significant and reversible increases in the receptive field size, spontaneous firing rate, response to brushing and pinching the skin and afterdischarge activity of dorsal horn neurones, as well as decreasing paired-pulse depression of responses evoked by transcutaneous electrical stimulation. In non-allodynic SCI animals, bicuculline ejection led to significant changes in receptive field size, paired-pulse depression and responses to brush and pinch stimulation that were comparable to those observed in normal animals. By contrast, in allodynic SCI animals, bicuculline ejection had little or no effect on dorsal horn neurone responses to mechanical skin stimuli and paired-pulse depression despite reliably blocking the inhibition of cell firing produced by similarly applied GABA. The demonstration of reduced GABAergic inhibition predominantly in the allodynic SCI rats suggests that such a deficiency contributed to this pain-related behaviour acutely following SCI.

Multiple metabotropic glutamate receptor subtypes modulate GABAergic neurotransmission in rat periaqueductal grey neurons in vitro.

The effect of metabotropic glutamate receptor (mGluR) activation on GABAergic synaptic transmission in rat periaqueductal grey (PAG) neurons was examined using whole-cell patch-clamp recordings in brain slices. The selective groups I, II and III mGluR agonists DHPG (10-30 microM), DCG-IV (1-3 microM) and L-AP4 (10-30 microM) inhibited electrically evoked GABA(A) mediated inhibitory postsynaptic currents (IPSCs) in all PAG neurons tested. DCG-IV and L-AP4 also reduced the frequency of spontaneous IPSCs, while DHPG produced both increases and decreases in spontaneous IPSC frequency in a dose dependent manner. In the presence of TTX, DHPG, DCG-IV and L-AP4 all reduced the frequency of spontaneous miniature IPSCs, but had no effect on their amplitudes. The DHPG, DCG-IV and L-AP4 effects on miniature IPSCs were dose dependent (EC(50)s=1.4, 0.055 and 0.52 microM, respectively) and were reduced by the selective mGluR antagonists MCPG, EGLU and MSOP, respectively. These results indicate that GABAergic synaptic transmission within the PAG is reduced by groups I, II and III mGluR activation via a presynaptic mechanism and is increased by group I mGluR activation via an action potential dependent mechanism. The finding of convergent groups I, II and III mGluR-mediated inhibition of synaptic transmission is novel and indicates that all groups of mGluRs have the potential to modulate the constellation of analgesic, behavioural and autonomic functions within the PAG.

Cellular actions of opioids on periaqueductal grey neurons from C57B16/J mice and mutant mice lacking MOR-1.

1 Patch clamp recordings were made from periaqueductal grey (PAG) neurons in vitro to investigate the cellular actions of opioids in wild-type C57B16/J mice and mutant mice lacking the first exon of the micro -opioid (MOP) receptor. 2 In wild-type mice, the kappa-(KOP) agonist U-69593 (300 nM) and the mixed micro /delta-opioid agonist met-enkephalin (10 micro M), but not the delta-(DOP) agonist deltorphin (300 nM), reduced the amplitude of evoked GABA(A)-mediated inhibitory postsynaptic currents (IPSCs). Met-enkephalin and U-69593 also reduced the rate of spontaneous miniature IPSCs, but had no effect on their amplitude and kinetics. In micro -receptor-deleted mice, only U-69593 (300 nM) reduced the amplitude of evoked IPSCs. 3 In wild-type mice, the MOP agonist DAMGO (3 micro M) produced an outward current in 76% of the neurons. Deltorphin and U-69593 produced outward currents in 24 and 32% of the neurons, respectively. In micro -receptor-deleted mice, deltorphin and U-69593 produced similar outward currents in 32 and 27% of the neurons, respectively, while DAMGO was without effect. All neurons in both the wild-type and micro -receptor-deleted mice responded with similar outward currents to either the GABA(B) receptor agonist baclofen (10 micro M), or the opioid-like receptor ORL1 (NOP) agonist nociceptin (300 nM). 4 The DAMGO-, deltorphin-, U-69593-, baclofen- and nociceptin-induced currents displayed inward rectification and reversed polarity at -109 to -116 mV. 5 These findings indicate that micro -, delta- and kappa-opioid receptor activation has complex pre- and postsynaptic actions within the mouse PAG. This differs to the rat PAG where only micro -opioid receptor actions have been observed.

Endocannabinoid modulation by FAAH and monoacylglycerol lipase within the analgesic circuitry of the periaqueductal grey.

BACKGROUND AND PURPOSE: Endogenous cannabinoids (endocannabinoids) in the periaqueductal grey (PAG) play a vital role in mediating stress-induced analgesia. This analgesic effect of endocannabinoids is enhanced by pharmacological inhibition of their degradative enzymes. However, the specific effects of endocannabinoids and the inhibitors of their degradation are largely unknown within this pain-modulating region. EXPERIMENTAL APPROACH: In vitro electrophysiological recordings were conducted from PAG neurons in rat midbrain slices. The effects of the major endocannabinoids and their degradation inhibitors on inhibitory GABAergic synaptic transmission were examined. KEY RESULTS: Exogenous application of the endocannabinoid, anandamide (AEA), but not 2-arachidonoylglycerol (2-AG), produced a reduction in inhibitory GABAergic transmission in PAG neurons. This AEA-induced suppression of inhibition was enhanced by the fatty acid amide hydrolase (FAAH) inhibitor, URB597, whereas a 2-AG-induced suppression of inhibition was unmasked by the monoacylglycerol lipase (MGL) inhibitor, JZL184. In addition, application of the CB1 receptor antagonist, AM251, facilitated the basal GABAergic transmission in the presence of URB597 and JZL184, which was further enhanced by the dual FAAH/MGL inhibitor, JZL195. CONCLUSIONS AND IMPLICATIONS: Our results indicate that AEA and 2-AG act via disinhibition within the PAG, a cellular action consistent with analgesia. These actions of AEA and 2-AG are tightly regulated by their respective degradative enzymes, FAAH and MGL. Furthermore, individual or combined inhibition of FAAH and/or MGL enhanced tonic disinhibition within the PAG. Therefore, the current findings support the therapeutic potential of FAAH and MGL inhibitors as a novel pharmacotherapy for pain.

Menthol enhances phasic and tonic GABAA receptor-mediated currents in midbrain periaqueductal grey neurons.

BACKGROUND AND PURPOSE: Menthol, a naturally occurring compound in the essential oil of mint leaves, is used for its medicinal, sensory and fragrant properties. Menthol acts via transient receptor potential (TRPM8 and TRPA1) channels and as a positive allosteric modulator of recombinant GABAA receptors. Here, we examined the actions of menthol on GABAA receptor-mediated currents in intact midbrain slices. EXPERIMENTAL APPROACH: Whole-cell voltage-clamp recordings were made from periaqueductal grey (PAG) neurons in midbrain slices from rats to determine the effects of menthol on GABAA receptor-mediated phasic IPSCs and tonic currents. KEY RESULTS: Menthol (150-750 µM) produced a concentration-dependent prolongation of spontaneous GABAA receptor-mediated IPSCs, but not non-NMDA receptor-mediated EPSCs throughout the PAG. Menthol actions were unaffected by TRPM8 and TRPA1 antagonists, tetrodotoxin and the benzodiazepine antagonist, flumazenil. Menthol also enhanced a tonic current, which was sensitive to the GABAA receptor antagonists, picrotoxin (100 µM), bicuculline (30 µM) and Zn(2+) (100 µM), but unaffected by gabazine (10 µM) and a GABAC receptor antagonist, 1,2,5,6-tetrahydropyridin-4-yl)methylphosphinic acid hydrate (TPMPA; 50 µM). In addition, menthol potentiated currents induced by the extrasynaptic GABAA receptor agonist THIP/gaboxadol (10 µM). CONCLUSIONS AND IMPLICATIONS: These results suggest that menthol positively modulates both synaptic and extrasynaptic populations of GABAA receptors in native PAG neurons. The development of agents that potentiate GABAA -mediated tonic currents and phasic IPSCs in a manner similar to menthol could provide a basis for novel GABAA -related pharmacotherapies.

Cholecystokinin exerts an effect via the endocannabinoid system to inhibit GABAergic transmission in midbrain periaqueductal gray.

Cholecystokinin modulates pain and anxiety via its functions within brain regions such as the midbrain periaqueductal gray (PAG). The aim of this study was to examine the cellular actions of cholecystokinin on PAG neurons. Whole-cell patch clamp recordings were made from rat midbrain PAG slices in vitro to examine the postsynaptic effects of cholecystokinin and its effects on synaptic transmission. Sulfated cholecystokinin-(26-33) (CCK-S, 100-300 nM), but not non-sulfated cholecystokinin-(26-33) (CCK-NS, 100-300 nM) produced an inward current in a sub-population of opioid sensitive and insensitive PAG neurons, which did not reverse over a range of membrane potentials. The CCK-S-induced current was abolished by the CCK1 selective antagonist devazepide (100 nM), but not by the CCK2 selective antagonists CI988 (100 nM, 1 µM) and LY225910 (1 µM). CCK-S, but not CCK-NS produced a reduction in the amplitude of evoked GABA(A)-mediated inhibitory postsynaptic currents (IPSCs) and an increase in the evoked IPSC paired-pulse ratio. By contrast, CCK-S had little effect on the rate and amplitude of TTX-resistant miniature IPSCs under basal conditions and when external K(+) was elevated. The CCK-S-induced inhibition of evoked IPSCs was abolished by the cannabinoid CB1 receptor antagonist AM251 (3 µM), the mGluR5 antagonist MPEP (10 µM) and the 1, 2-diacylglycerol lipase (DAGLα) inhibitor tetrahydrolipstatin (10 µM). In addition, CCK-S produced an increase in the rate of spontaneous non-NMDA-mediated, TTX-dependent excitatory postsynaptic currents (EPSCs). These results suggest that cholecystokinin produces direct neuronal depolarisation via CCK1 receptors and inhibits GABAergic synaptic transmission via action potential-dependent release of glutamate and mGluR5-induced endocannabinoid signaling. Thus, cholecystokinin has cellular actions within the PAG that can both oppose and reinforce opioid and cannabinoid modulation of pain and anxiety within this brain structure.