Nicotinic modulation of descending pain control circuitry - PubMed (original) (raw)

Nicotinic modulation of descending pain control circuitry

Iboro C Umana et al. Pain. 2017 Oct.

Abstract

Along with the well-known rewarding effects, activation of nicotinic acetylcholine receptors (nAChRs) can also relieve pain, and some nicotinic agonists have analgesic efficacy similar to opioids. A major target of analgesic drugs is the descending pain modulatory pathway, including the ventrolateral periaqueductal gray (vlPAG) and the rostral ventromedial medulla (RVM). Although activating nAChRs within this circuitry can be analgesic, little is known about the subunit composition and cellular effects of these receptors, particularly within the vlPAG. Using electrophysiology in brain slices from adult male rats, we examined nAChR effects on vlPAG neurons that project to the RVM. We found that 63% of PAG-RVM projection neurons expressed functional nAChRs, which were exclusively of the α7-subtype. Interestingly, the neurons that express α7 nAChRs were largely nonoverlapping with those expressing μ-opioid receptors (MOR). As nAChRs are excitatory and MORs are inhibitory, these data suggest distinct roles for these neuronal classes in pain modulation. Along with direct excitation, we also found that presynaptic nAChRs enhanced GABAergic release preferentially onto neurons that lacked α7 nAChRs. In addition, presynaptic nAChRs enhanced glutamatergic inputs onto all PAG-RVM projection neuron classes to a similar extent. In behavioral testing, both systemic and intra-vlPAG administration of the α7 nAChR-selective agonist, PHA-543,613, was antinociceptive in the formalin assay. Furthermore, intra-vlPAG α7 antagonist pretreatment blocked PHA-543,613-induced antinociception via either administration method. Systemic administration of submaximal doses of the α7 agonist and morphine produced additive antinociceptive effects. Together, our findings indicate that the vlPAG is a key site of action for α7 nAChR-mediated antinociception.

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Conflict of interest statement

The authors have no financial conflicts of interest related to the results of this manuscript.

Figures

Figure 1

Figure 1. A subpopulation of PAG-RVM projection neurons expresses α7 nAChRs

A. Left: Fluorescence photomicrograph illustrating a sample RVM injection site (−10.5 mm from Bregma). Gigantocellular Reticular Nucleus (GiA) and Raphe Magnus Nucleus (RMg) are part of the RVM. (Scale: 500 um). Right: Example vlPAG backlabeled neuron. (Scale: 20 µm). B. Representative fluorescence images of a backlabeled vlPAG neuron that projects to the RVM (left), antibody staining for tryptophan hydroxylase (TPH), a marker of serotonergic neurons (center), and image overlay showing no co-localization of the fluorescent signals (right; scale = 100 µm). We saw no overlap with TPH immunofluorescence in 77 backlabeled cells. C. Inward current response to a brief focal application of acetylcholine (ACh; 3 mM; 300 msec duration). Similar responses were seen in 49 of 78 backlabeled neurons tested. D. Inward current response to focal application of the selective α7 agonist, PHA-543613 (100 µM; 300 msec). Similar responses were seen in 8/16 neurons tested. E. Focal ACh application (3mM) induced an inward current with similar kinetics whether the application was 300 msec (black trace) or 2 sec (light grey trace) in duration. Bath application of MLA (10 nM) completely blocked the ACh-induced current in the same neuron. Complete blockade by MLA was seen in all responsive cells tested (n=15). F. Summary of response prevalence of vlPAG neurons to ACh alone and in combination with MLA (10 nM; tested only on responsive cells) or αBGT (50 nM; pretreated for >15 min prior to recording); n values are presented within or above each bar. G. Inward current due to focal application of ACh (3 mM). H. Inward current response to ACh (in the same neuron as G.) after treatment with the α7 positive allosteric modulator, PNU-120596 for15 min. The prolonged inward current is consistent with loss of α7 nAChR desensitization due to PNU-120596.

Figure 2

Figure 2. Functional nAChR and MOR expression distinguishes 2 major functional subclasses of PAG-RVM projection neurons

Functional nAChR response plotted vs. MOR response magnitudes for 37 PAG-RVM projection neurons tested for responses to both ACh (3 mM; top inset) and endomorphin-1 (EM-1; 1 µM; bottom inset). Each symbol on the graph represents the magnitude of the response to focal ACh (y-axis) plotted against the magnitude EM-1 responses (x-axis) for each neuron. The filled circles represent those neurons with measurable responses to only one agonist, and the open circles represent the neurons that responded to both agonists. Note that the open triangle 0/0 data point represents 9 neurons that showed no response to either agonist.

Figure 3

Figure 3. Non-α7 nAChRs enhance excitatory and inhibitory drive to α7-expressing and α7-lacking vlPAG neurons

A. Example mEPSC raw traces recorded in the presence of TTX and bicuculline (Bic) during baseline, nicotine (1 µM) application, and washout. Example mEPSC frequency histogram from an α7-expressing neuron. B. Prevalence of nicotine-induced increase in EPSC frequency among α7-expressing and α7-lacking vlPAG neurons. Although nicotine enhances excitatory inputs to vlPAG projection neurons, the fraction of neurons that responded to nicotine with increases in either mEPSC or sEPSC frequency was nearly identical between α7-expressing (3/6, 6/11; white bars) and α7-lacking neurons (3/6, 6/10; gray bars). C. Summary of average nicotine-induced increases in EPSC frequency. Individual measurements are illustrated by the symbol scatterplots. There were no differences between groups. D. Summary of nicotine effects on average EPSC amplitudes for all groups. Nicotine did not alter EPSC amplitudes in any of the groups. E. Example mIPSC raw traces recorded in the presence of DNQX and TTX during baseline, nicotine application, and washout. Example sIPSC frequency histogram from an α7-lacking neuron. F. The fraction of neurons that responded to nicotine with enhanced mIPSC and sIPSC frequency was higher in α7-lacking neurons (5/9, 12/19; gray bars) than in α7-expressing neurons (2/10, 7/24; white bars) * p < 0.05. G. Summary of nicotine-induced changes in IPSC frequency. The α7-expressing and α7-lacking neurons showed similar average increases in IPSC frequency. H. There was no effect of nicotine on IPSC amplitude in any of the groups (bars represent Mean ± S.E.M of frequency/amplitude).

Figure 4

Figure 4. Intra-vlPAG administration of α7 nAChR agonist and PAM produces antinociception in the formalin assay

A. (Top) Timeline of behavioral experiments. (Bottom) Time course of nociceptive mean scores following intraplantar injection of formalin in the left hindpaw. The curves illustrate responses during Phase I (0–10 min), interphase (10–15 min) and Phase II (15–60 min). Rats received focal injections into the vlPAG of either DAMGO (1.9 nmol, filled diamonds, n=8), PNU-120596 (0.9 nmol, inverted, filled triangles, n=8), or vehicle (open circles, n=13) followed 10 min later by the intraplantar formalin injection. The PHA-543613 infusion (3 nmol, open triangles, n=8) was administered 5 min before formalin injection. Each symbol represents the nociceptive mean score ± S.E.M, representing the time spent displaying nocifensive behavior during 5 min periods. B. Summary of nociceptive duration in the tonic phase (Phase II). Each bar represents the mean ± S.E.M. of the total time spent exhibiting nocifensive behaviors during Phase II (15–60 min after formalin injection). The shading within each bar illustrates the proportion of time favoring (white), lifting (gray), or licking (black). Holm-Sidak’s multiple comparisons test (relative to vehicle): ** p<0.01, **** p<0.0001. C. Summary of mean nociceptive duration for vehicle and two different concentrations of PHA-543613 (0.3 nmol n = 4; 3 nmol n = 8; **** p<0.0001) administered into the vlPAG. D. Duration of nociceptive responses during Phase II of the formalin test (y-axis) plotted versus the position of the injections of PHA-543613 (3 nmol) lateral to the vlPAG (x-axis). Each filled circle corresponds to an individual rat. E. Summary of mean nociceptive duration for vehicle and three different concentrations of PNU-120596 (9 pmol: n =4; 90 pmol: n = 5; ** p<0.01) administered into the vlPAG. F. Post-experiment determination of drug injection sites with fluorescent dye injections. Only those rats with dye located within the vlPAG were included in this study. Diamonds: DAMGO; Triangles: PHA-543613; Squares: PNU-120596, Circles: vehicle.

Figure 5

Figure 5. Intra-vlPAG administration of α7 nAChR antagonists block PHA-543613 mediated antinociception

A. Time course of the nocifensive responses during the formalin test. To assess whether the effects of focal PHA-543613 infusion were mediated by α7 nAChR activation in the vlPAG, rats received intra-vlPAG injections of αBGT (1.5 pmol, inverted, filled triangles, n=6) or MLA (90 pmol, filled diamonds, n=6), followed by PHA-543613 (3 nmol). Pretreatment with αBGT or MLA was 30 or 10 min before formalin injection, respectively. PHA-543613 was focally infused 5 min before formalin injection. Responses to vehicle (circles) and PHA-543613 alone (triangles) from Figure 4 are included for comparison. For clarity, the time course of nocifensive responses to MLA and αBGT were not included in this graph. B. Mean duration of Phase II nocifensive behavior. Pretreatment with either MLA or αBGT completely blocked the antinociceptive effects of PHA-543613 during Phase II of the formalin test (Holm-Sidak’s multiple comparisons test: *** p<0.001). The shading within each bar illustrates the proportion of time favoring (white), lifting (gray), or licking (black). Pretreatment with antagonists alone (MLA: n=6; and αBGT: n=5) showed no difference relative to vehicle.

Figure 6

Figure 6. Antinociceptive effect of systemic α7 nAChR agonist administration is blocked by intra-vlPAG administration of an α7 antagonist

A. Nociceptive behaviors plotted versus time following formalin injection. Systemic PHA-543613 alone (4 mg/kg s.c., n=5, open triangles) resulted in antinociceptive effects during Phase II of the formalin test. To test the contribution of α7 nAChRs in the vlPAG, αBGT (1.5 pmol) was focally administered into the vlPAG 30 min prior to formalin injection. Rats were then injected with PHA-543613 (4 mg/kg; s.c., n=5) followed by intraplantar formalin injection 5 min later (filled squares). αBGT completely blocked PHA-543613 antinociception and resulted in nocifensive responses similar to baseline (vehicle, s.c., n=7, open circles). B. Mean duration of Phase II nociceptive behavior. A range of PHA-543613 doses (0.2 mg/kg: n=7; 1 mg/kg: n=5; 2 mg/kg: n=5; 10 mg/kg: n=4) was tested. Intra-vlPAG αBGT completely blocked the antinociceptive effects of PHA-543613 (4 mg/kg) during Phase II of the formalin test (Holm-Sidak’s multiple comparison test: *** p < 0.001, ** p < 0.01). The shading within each bar illustrates the proportion of time favoring (white), lifting (gray), or licking (black).

Figure 7

Figure 7. Co-administration of low-dose α7 nAChR and morphine produce PAG-dependent antinociception

A. Nociceptive behaviors plotted versus time following formalin injection. Systemic pretreatment of morphine (4 mg/kg, open triangles; 0.67 mg/kg, filled triangles; 0.067 mg/kg, filled diamonds) or vehicle (open circles) reveals a concentration-dependent decrease in nociceptive responses with complete block of Phase II responses after treatment with the highest morphine dose. B. Dose-effect relationship for morphine vs the duration of Phase II nocifensive responses. The shading within each bar illustrates the proportion of time favoring (white), lifting (gray), or licking (black). (Vehicle; n = 11; Morphine 4 mg/kg, n=4; 2 mg/kg, n=7; 0.67 mg/kg, n=6; or 0.067 mg/kg, n=4) Holm-Sidak’s multiple comparison test: **** p < 0.0001 C. Bar graph of mean duration of Phase II nociceptive behavior. Weak antinociceptive effects were seen with systemic administration of intermediate doses of morphine (0.67 mg/kg) or PHA-543613 (2 mg/kg) alone. The combination of these drugs (n=7) resulted in significant antinociception when compared with vehicle (Holm-Sidak’s multiple comparison test: ** p < 0.01). Intra-vlPAG αBGT completely blocked antinociceptive effect of morphine/PHA-543613 co-administration (n=4). The shading within each bar illustrates the proportion of time favoring (white), lifting (gray), or licking (black). Holm-Sidak’s multiple comparison test: * p<0.05. D. Dose-response profile of systemic administration of PHA-543613 in the absence (filled circles) or in combination with morphine (0.67 mg/kg; open circles) administered 20 min before formalin injection. A range of PHA-543613 doses was tested in combination with morphine: 1 mg/kg: n=4; 2 mg/kg: n=7; 4 mg/kg: n=4). Filled square indicates vehicle injection (0 mg/kg PHA-543613). Holm-Sidak’s multiple comparison test: *** p < 0.001, **** p < 0.0001.

Figure 8

Figure 8. Model of α7 nAChR-mediated analgesia in the vlPAG

Our experiments show that vlPAG α7 activation produces robust antinociception principally through the activation of somatic receptors on a subset of neurons within that nucleus. Our behavioral data support the idea that pain inhibitory neurons in the vlPAG suppress ascending nociceptive signaling through connections in the RVM to modulate excitability in the spinal cord dorsal horn. MOR agonist application inhibits a separate group of vlPAG projection neurons that facilitate nociceptive signaling.

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