Neurotensin inhibition of GABAergic transmission via mGluR-induced endocannabinoid signalling in rat periaqueductal grey

Abstract

Neurotensin modulates pain via its actions within descending analgesic pathways which include brain regions such as the midbrain periaqueductal grey (PAG). The aim of this study was to examine the cellular actions of neurotensin on PAG neurons. Whole cell patch clamp recordings were made from rat midbrain PAG slices in vitro to examine the postsynaptic effects of neurotensin and its effects on GABA(A) mediated inhibitory postsynaptic currents (IPSCs). Neurotensin (100-300 nM) produced an inward current in subpopulations of opioid sensitive and insensitive PAG neurons which did not reverse over membrane potentials between -50 and -130 mV. The neurotensin induced current was abolished by the NTS1 and NTS1/2 antagonists SR48692 (300 nM) and SR142948A (300 nM). Neurotensin also produced a reduction in the amplitude of evoked IPSCs, but had no effect on the rate and amplitude of TTX-resistant miniature IPSCs. The neurotensin induced inhibition of evoked IPSCs was reduced by the mGluR5 antagonist MPEP (5microM) and abolished by the cannabinoid CB(1) receptor antagonist AM251 (3 microM). These results suggest that neurotensin produces direct neuronal depolarisation via NTS1 receptors and inhibits GABAergic synaptic transmission within the PAG. The inhibition of synaptic transmission is mediated by neuronal excitation and action potential dependent release of glutamate, leading to mGluR5 mediated production of endocannabinoids which activate presynaptic CB(1) receptors. Thus, neurotensin has cellular actions within the PAG which are consistent with both algesic and analgesic activity, some of which are mediated via the endocannabinoid system.

Figure 1. Neurotensin produces an inward current in subpopulations of μ-opioid sensitive and insensitive neurons

A–D, current traces of neurotensin sensitive (A and B) and insensitive (C and D) PAG neurons during superfusion of neurotensin (NT, 300 nm), met-enkephalin (ME, 10 μm), baclofen (10 μm) and CGP55845 (CGP, 1μm). E, bar chart displaying the percentage of met-enkephalin responding (ME +ve) and met-enkephalin non-responding (ME –ve) neurons that responded to neurotensin and baclofen. F, the current–voltage relationship for a PAG neuron in which neurotensin produced an inward current. Membrane currents were evoked by voltage steps in 10 mV increments from –50 mV to –130 mV (250 ms duration). Scale bars in A–B are 10 pA and 1 min. A, B, C, D and F are from different neurons.

Figure 2. The actions of neurotensin are mediated by NTS1 and possibly NTS2 receptors

A and B, current traces during two consecutive applications of neurotensin (NT, 100 nm) and then baclofen (10 μm), in which either the NTS1 antagonist SR48692 (300 nm) (A), or the NTS1/2 antagonist SR142948A (300 nm) (B) was added following the first washout of neurotensin. C, bar chart showing the inward currents produced during two consecutive applications of neurotensin (NT 1st and 2nd), in which either no antagonist (Control), SR48692 (300 nm), or SR142948A (300 nm) was applied during the second application of neurotensin. **P < 0.01. Scale bars in A–B are 10 pA and 2 min.

Figure 3. Neurotensin inhibits evoked IPSCs

A, time course of evoked IPSC amplitude (eIPSC Ampl) during application of neurotensin (NT, 100 nm), baclofen (10 μm) and CGP55845 (CGP, 1 μm). B, averaged evoked IPSCs before (Pre) and during application of neurotensin and baclofen. C, averaged evoked IPSCs in response to identical paired stimuli (inter-stimulus interval = 70 ms) for the traces in B, with IPSC₁ normalized to demonstrate relative facilitation of IPSC₂ during superfusion of neurotensin. D, scatter plot showing the amplitude of the first evoked IPSC (eIPSC1) and the ratio of evoked IPSC₂/IPSC₁ (eIPSC2:1) in the presence of neurotensin (100 nm) expressed as a percentage of the pre-neurotensin (pre-NT) level. *P < 0.05, ***P < 0.001. Traces in A–C are from the same neuron.

Figure 4. Neurotensin has no effect on miniature IPSCs

A, time course of miniature IPSC (mIPSC) rate during superfusion of neurotensin (NT, 300 nm) and met-enkephalin (ME, 10 μm). B, raw current traces of miniature IPSCs before (Pre) and during superfusion of neurotensin and met-enkephalin. C and D, cumulative probability distribution plots of miniature IPSC inter-event interval (C) and amplitude (D), before and during superfusion of neurotensin and met-enkephalin. E, averaged traces of miniature IPSCs before and during superfusion of neurotensin and met-enkephalin, for the corresponding epoch in (C) and (D). F, bar chart of the mean rate and amplitude of miniature IPSCs in the presence of neurotensin and met-enkephalin in both normal (2.5 mm) and high K⁺ (17.5 mm) containing ACSF, expressed as a percentage of the pre-drug level. **P < 0.01, ***P < 0.001. Traces in A–E are from the same neuron.

Figure 5. Neurotensin inhibition of evoked IPSCs is mediated by mGluR5 induced endocannabinoid signalling

A–C, averaged evoked IPSCs before (Pre) and during neurotensin (NT, 300 nm) and baclofen (10 μm) in slices pre-incubated in the mGluR5 antagonist MPEP (5 μm) (A), the CB₁ antagonist AM251 (3 μm) (B), or the TRPV1 antagonist capsazepine (Capsaz, 10 μm) (C). D, bar chart showing the percentage inhibition of evoked IPSCs produced by neurotensin (300 nm) alone (Ctl), and in the presence of MPEP (5 μm), AM251 (3 μm), or capsazepine (10 μm). *P < 0.05, ***P < 0.001. Traces in A–C are from different neurons, with scale bars of 100 pA and 10 ms.