Substance P Drives Endocannabinoid-Mediated Disinhibition in a Midbrain Descending Analgesic Pathway

Abstract

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 , ,␦-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.

Cholecystokinin Exerts an Effect via the Endocannabinoid System to Inhibit GABAergic Transmission in Midbrain Periaqueductal Gray

“…The lack of effect of cholecystokinin on TTX-resistant miniature IPSCs suggests that the cholecystokinin-induced reduction in presynaptic GABA release involved additional mechanisms external to the synapse, rather than direct postsynaptic CCK1 receptorinduced retrograde endocannabinoid signaling, as observed for mGluR5 and M1 mAChR receptor signaling in PAG (Drew et al, 2008;Lau and Vaughan, 2008). We have previously shown that activation of G q -coupled NK1 and NTS1/2 receptors indirectly activates the mGluR5-endocannabinoid system by enhancing endogenous glutamate release (Drew et al, 2009;Mitchell et al, 2009). In this study, CCK-S produced neuronal depolarization and an increase in spontaneous action potential-dependent glutamatergic EPSCs, however, we cannot rule out a direct CCK receptor-mediated enhancement of presynaptic glutamate release (Yang et al, 2006).…”

“…These findings suggest that the cholecystokinin-induced inhibition of evoked IPSCs is also mediated by the endocannabinoid system, because it was abolished by the cannabinoid CB1 antagonist/inverse agonist AM251. Furthermore, the endocannabinoid involved in this process was likely to be 2-arachidonyl glycerol because the CCK-Sinduced inhibition was blocked by an inhibitor of DAGLa, the enzyme which mediates its production via Gq-coupled GPCRs in a number of brain regions, including the PAG (Bisogno et al, 2003;Drew et al, 2009;Jung et al, 2005;Lau and Vaughan, 2008;Uchigashima et al, 2007).…”

“…All agents were diluted to working concentrations in ACSF (solvent p0.03% v/v) immediately before use and applied by superfusion. Cholecystokinin, cannabinoid, and mGluR agents were used at concentrations that have previously been shown to be maximally effective in PAG slices (Drew et al, 2009;Drew et al, 2008;Yang et al, 2007). The entire system was washed in 30% ethanol after AM251 experiments.…”

“…which engages mGluR5-induced endocannabinoid signaling (Drew et al, 2009;Mitchell et al, 2009). To address this, we first examined whether CCK-S increased action potential-dependent release of glutamate.…”

“…Although functional studies have shown that the GABAergic system has a key role in the modulation of pain and anxiety within the PAG (Fields et al, 2006;Graeff et al, 1993), the effect of cholecystokinin on GABAergic synaptic transmission within this brain structure is unknown. We have recently demonstrated that activation of other G q -coupled receptors, including metabotropic glutamate (mGluR5), muscarinic (M1 mAChR), neurokinin (NK1/2), and neurotensin (NTS1/2) receptors, indirectly inhibits GABAergic synaptic transmission via the endocannabinoid system within the PAG (Drew et al, 2008(Drew et al, , 2009Lau and Vaughan, 2008;Mitchell et al, 2009). In this regard, cannabinoid agonists and endocannabinoids modulate analgesic and anxiolytic activity within the PAG, including a component of stress-induced analgesia (Finn et al, 2003;Hohmann et al, 2005;Lichtman et al, 1996;Moreira et al, 2007).…”