Geoffrey M Drew

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

&NA; 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 &ggr;‐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 GABAA 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.

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-β-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 μm), 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.

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.

Inhibition of fatty acid amide hydrolase unmasks CB1 receptor and TRPV1 channel-mediated modulation of glutamatergic synaptic transmission in midbrain periaqueductal grey.

BACKGROUND AND PURPOSE While arachidonyl ethanolamine (anandamide) produces pharmacological effects mediated by cannabinoid CB1 receptors, it is also an agonist at the transient receptor potential vanilloid type 1 (TRPV1) ion channel. This study examined the cellular actions of anandamide in the midbrain periaqueductal grey (PAG), a region implicated in the analgesic actions of cannabinoids, and which expresses both CB1 receptors and TRPV1.

Cellular actions of opioids on periaqueductal grey neurons from C57B16/J mice and mutant mice lacking MOR-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 μ‐opioid (MOP) receptor. In wild‐type mice, the κ‐(KOP) agonist U‐69593 (300 nM) and the mixed μ/δ‐opioid agonist met‐enkephalin (10 μM), but not the δ‐(DOP) agonist deltorphin (300 nM), reduced the amplitude of evoked GABAA‐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 μ‐receptor‐deleted mice, only U‐69593 (300 nM) reduced the amplitude of evoked IPSCs. In wild‐type mice, the MOP agonist DAMGO (3 μM) 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 μ‐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 μ‐receptor‐deleted mice responded with similar outward currents to either the GABAB receptor agonist baclofen (10 μM), or the opioid‐like receptor ORL1 (NOP) agonist nociceptin (300 nM). The DAMGO‐, deltorphin‐, U‐69593‐, baclofen‐ and nociceptin‐induced currents displayed inward rectification and reversed polarity at −109 to −116 mV. These findings indicate that μ‐, δ‐ and κ‐opioid receptor activation has complex pre‐ and postsynaptic actions within the mouse PAG. This differs to the rat PAG where only μ‐opioid receptor actions have been observed.

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, GABAB, μ,δ-opioid, adenosine A1, and 5HT1A 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.

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.

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

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.

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 GABAA-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.

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

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.