Background & Aims
The central nucleus of the amygdala (CeA) is a key region in processing nociceptive inputs1 and modulating sensory and affective dimensions of pain2,3. The CeA consists of a heterogeneous population of neurons4. One major subclass of CeA neurons expresses corticotropin-releasing hormone (CRH; CeA-CRH neurons)5 that are innervated by the parabrachial nucleus (PBn)6,7, a key relay node in the ascending pain pathway. Therefore, CeA-CRH neurons likely play a significant role in pain processing, but mechanisms by which pain drives changes to circuit and synaptic function of CeA-CRH neurons remain unclear. Here we hypothesized the bioactive sphingolipid sphingosine-1-phosphate (S1P) regulates CeA-CRH circuits in a neuropathic pain model8-10. Our goals were to measure intrinsic and synaptic alterations to CeA-CRH neurons in the spared-nerve injury (SNI) model of neuropathic pain and determine if S1P receptor 1 (S1PR1) agonist SEW2871 can reverse any significant changes induced by SNI.
Methods
CRH (B6(Cg)-Crhtm1(cre)Zjh/J) mice were mated to tdTomato (B6.Cg-Gt(ROSA)26Sortm9(CAG-tdTomato)Hze/J) mice (Jackson) resulting in offspring with red fluorescence in CeA-CRH neurons. For SNI, the common peroneal and tibial branches of the sciatic nerve were severed, leaving the sural branch intact. Mechanical allodynia was assessed via Von Frey filaments. On post-operative day 14 (POD14), acute coronal brain slices (300µm) were prepared and incubated in artificial cerebrospinal fluid (aCSF): 37°C for 30 minutes, 21-22°C for 60 minutes. Slices were transferred to a recording chamber of a SliceScopePro 6000 (Scientifica). Whole-cell patch-clamp recordings from fluorescently labeled CeA-CRH neurons in acute slice were amplified and filtered at 4 kHz and digitized at 10 kHz using a Multiclamp 700B amplifier (Molecular Devices). 100nM of SEW2871 (S1PR1 agonist, Cayman Chemical) dissolved in DMSO was added directly to the aCSF at least 15 minutes prior to electrophysiological recordings.
Results
CRH-tdTomato mice demonstrated significant mechanical allodynia following nerve injury compared to sham littermates. At POD14, CeA-CRH neurons recorded from SNI mice exhibit lower current and voltage thresholds for action potential (AP) firing, while exhibiting no significant change in firing frequency. Surprisingly, treatment with SEW2871 had no significant effect on firing frequency or threshold for AP firing in CeA-CRH neurons from either sham or SNI mice. At POD14, CeA-CRH neurons recorded from sham and SNI mice of both sexes showed no difference in amplitude of spontaneous excitatory postsynaptic currents (sEPSCs), which was also unaffected by addition of SEW2871. However, CeA-CRH neurons recorded from SNI male mice displayed an increase in sEPSC frequency compared to sham male mice. Interestingly, this increase in sEPSC frequency was not observed when CeA-CRH neurons were incubated with SEW2871. Analyses of preliminary data in females currently shows no significant differences.
Conclusions
Our results indicate that SNI sensitizes CeA-CRH neurons via decreased threshold for AP firing and enhanced frequency of glutamatergic inputs. Enhanced frequency of sEPSCs in CeA-CRH neurons is currently observed in male mice, but not female mice suggesting a sex-specific mechanism for SNI-induced sensitization of CeA-CRH neurons. While treatment with S1PR1 agonist SEW2871 did not significantly alter intrinsic properties of CeA-CRH neurons, it did reduce an SNI-induced increase in sEPSC frequency, indicating the involvement of S1P in presynaptic mechanisms for glutamate release, which may be relevant to pain. Future experiments will dissect pain-induced and S1P-induced changes to distinct synaptic inputs onto CeA-CRH neurons.
References
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Presenting Author
Regan C. Hines
Poster Authors
Topics
- Models: Chronic Pain - Neuropathic