Analysis of basal neurotransmission and properties of spinal-PAG projection neurons in spinal slices derived from SNS-PKG-I^−/− mice and their PKG-I^fl/fl littermates.

<p>(A, B) Short-term depression during the conditioning train of low-frequency stimulation (A) and input-output curves for basal synaptic transmission between C-fibers and spinal-PAG projections neurons (B) were not different between PKG-I^fl/fl mice and SNS-PKG-I^−/− mice (<i>n</i> = 12 each). (C, D) Activation properties of spinal neurons are not altered in SNS-PKG-I^−/− mice as compared to their PKG-I^fl/fl littermates. Dorsal root stimulation threshold for evoking action potentials (C) in spinal-PAG projection neurons and spiking properties of spinal-PAG projection neurons upon direct current injection (D) are similar in SNS-PKG-I^−/− mice and their PKG-I^fl/fl littermates. (E) Resting membrane potential (RMP), action potential (AP) properties, such as threshold, amplitude, half-width, and the latency of first AP, and amplitude of after hyperpolarization (AHP) were similar between genotypes (<i>p</i>>0.05; Student's <i>t</i> test). (F, G) Typical traces (F) and magnitude of C-fiber volleys in L4/L5 roots recorded at different intensities of stimulation are comparable in SNS-PKG-I^−/− mice and their PKG-I^fl/fl littermates (<i>n</i> = 16 each).</p

Nociceptor-specific deletion of PKG-I and lack of developmental defects in SNS-PKG-I^−/− mice.

<p>(A) Immunoreactivity for an anti-PKG-I antibody was detected widely in DRG neurons of mice carrying floxed PKG-I alleles (PKG-I^fl/fl) and was lost either selectively in SNS-PKG-I^−/− mice or entirely in global PKG-I^−/− mice. (B) Quantitative size analysis of neurons of the dorsal root ganglia (DRG) expressing PKG-I in SNS-PKG-I^−/− mice and PKG-I^fl/fl littermates showing specific loss of PKG-I in small-diameter neurons in SNS-PKG-I^−/− mice. Data in panels represent mean ± S.E.M.; <i>n</i> = 10–15 DRG sections each. (C, D) Typical examples (C) and quantitative summary (D) from dual immunofluorescence experiments showing that in SNS-PKG-I^−/− mice, PKG-I immunoreactivity is abrogated from nociceptors, i.e. Isolectin-B₄-positive (IB₄) or Substance P-positive (α-Sub P) neurons, but retained in neurofilament 200-positive (α-NF200) large diameter neurons, in comparison with control littermates (PKG-I^fl/fl). <i>n</i> = 10–15 DRG sections each; * <i>p</i><0.001, ANOVA, post hoc Fisher's test. (E) Typical examples of anti-PKG-I immunostaining in the brain (cerebellar Purkinje neurons are shown) and spinal dorsal horns of SNS-PKG-I^−/− mice, their PKG-I^fl/fl littermates, and global PKG-I^−/− mice. Spinal dorsal horns of SNS-PKG-I^−/− mice showed a loss of anti-PKG-I immunoreactivity in the superficial neuropil (+), but distinct preservation of signals in cell bodies (arrowheads). (F) Typical examples of patterning of Trk-A-expressing sensory afferents (arrowheads in upper panels) and T-branching of DiI-labelled sensory afferents (arrows in lower panels) in the spinal cord in PKG-I^fl/fl mice and SNS-PKG-I^−/− mice at embryonic day 13.5 (E13.5). Scale bars represent 50 µm in panel A, 50 µm in panels A and C, 100 µm for spinal cord and 50 µm for cerebellum in panel E, and 100 µm in panel F. DRG, dorsal root ganglia; S.C., spinal cord.</p

PKG-I mediates nociceptive activity-dependent phosphorylation of Inositol 1,4,5-triphosphate receptor 1 (IP₃R1) in DRG and potentiates evoked calcium transients.

<p>(A, B) Typical examples (A) and quantitative summary (B) of levels of immunoprecipitated IP₃R1 phosphorylated at serine 1755 or total IP₃R1 in L4-L5 DRG lysates derived from naïve or formalin-injected PKG-I^fl/fl and SNS-PKG-I^−/− mice. (C–E) Comparison of evoked calcium transients in DRG neurons in culture derived from SNS-PKG-I^−/− mice or their control PKG-I^fl/fl littermates. (c) Typical examples of Fura2-loaded dissociated DRG neurons, which were co-stained with Fluor 488-conjugated isolectin B4 (IB4) to identify non-peptidergic nociceptive neurons (arrowheads). (D) Typical examples of traces of calcium transients evoked by bath application of bradykinin (BK, 50 nM), UTP (100 µM), KCl (25 mM), and capsaicin (1 µM) in IB4- and Fura2-double-labelled DRG neurons from PKG-I^fl/fl and SNS-PKG-I^−/− mice. (E) Ratiometric Fura2-based imaging of calcium released upon bath application of bradykinin (BK, 50 nM), UTP (100 µM), KCl (25 mM), and capsaicin (1 µM) in IB4- and Fura2-double-labelled nociceptive DRG neurons from PKG-I^fl/fl and SNS-PKG-I^−/− mice (9–10 independent culture experiments). Note that UTP-induced transients in non-nociceptive large-diameter neurons (e.g., arrow in panel C) are not different across genotypes. * <i>p</i><0.05, ANOVA of random measures followed by post hoc Fisher's test. Scale bar represents 20 µm in panel C.</p

Analysis of a presynaptic component to C-fiber-evoked LTP on spinal-PAG projection neurons.

<p>(A, B) Activation of C-nociceptors potentiates synaptic transmission by decreasing the rate of synaptic failures via activation of presynaptic PKG-I. Shown are typical examples of the frequency of synaptic failures (arrows) and synaptic successes (C-fiber-evoked EPSCs) evoked by minimal stimulation of dorsal roots prior to and after application of the conditioning stimulus in PKG-I^fl/fl mice (A) and SNS-PKG-I^−/− mice (B). (C) Summary of average failure rates upon minimal stimulation of dorsal roots prior to and 30 min following the conditioning stimulus (<i>n</i> = 5 slices for PKG-I^fl/fl mice and 7 slices for SNS-PKG-I^−/− mice). (D–F) Analysis of paired-pulse facilitation (PPF) and paired-pulse depression (PPD) of C-fiber-evoked EPSCs induced by pairs of stimuli at inter-stimulus interval of 110 ms in spino-PAG projection neurons from PKG-I^fl/fl and SNS-PKG-I^−/− mice. Traces of typical recordings showing PPF or PPD prior to (basal) and at 30 min following low-frequency conditioning stimulation of C-fibers (LFS) are shown in panel D. Paired-pulse ratios (PPR) prior to and at 30 min after conditioning stimulation are plotted in panel E (PKG-I^fl/fl mice) and panel F (SNS-PKG-I^−/− mice). Values of PPR above and below 1 prior to conditioning stimulation are represented as PPF (pink field) and PPD (blue field), respectively. Note that in PKG-I^fl/fl mice, neurons showing low initial PPF show an increase in PPF after conditioning stimulation, whereas neurons showing an initial high PPF show a decrease in PPF following conditioning stimulation, which corresponds to an increase in probability of release. These changes are reduced or do not come about in SNS-PKG-I^−/− mice. All data are represented as mean ± S.E.M. * <i>p</i><0.05, ANOVA of random measures followed by post hoc Fisher's test.</p

Rescue of defects in plasticity mechanisms in SNS-PKG-I^−/− mice by viral expression of GFP-tagged PKG-I specifically in the L3-L4 dorsal root ganglia (DRG) of adult mice.

<p>(A) Photomicrographs showing expression of GFP-tagged PKG-I in the L4 DRG upon delivery via adeno-associated virions (AAV). Scale bar represents 250 µm. (B) Basal mechanical sensitivity to graded mechanical stimuli (von Frey) is unchanged upon overexpression of GFP-tagged PKG-I or GFP alone (control) in DRG neurons of PKG-I^fl/fl mice and SNS-PKG-I^−/− mice. (C) PKG-I^fl/fl mice expressing GFP-tagged PKG-I show a minor increase in magnitude of capsaicin-induced mechanical hypersensitivity than PKG-I^fl/fl mice expressing GFP. † <i>p</i><0.05 as compared to AAV-GFP-PKG-I^fl/fl mice. (D) SNS-PKG-I^−/− mice overexpressing GFP in DRG show markedly reduced mechanical hypersensitivity with capsaicin than PKG-I^fl/fl mice overexpressing GFP. Overexpression of GFP-tagged PKG-I fully restored mechanical hypersensitivity in SNS-PKG-I^−/− mice. † <i>p</i><0.05 indicates significant differences in the AAV-GFP-SNS-PKG-I^−/− mice as compared to the other two groups. ANOVA followed by post hoc Fisher's test.</p

Nociceptive activity-driven, PKG-I-dependent phosphorylation of myosin light chains (MLC) in DRG in vivo and its contribution to synaptic potentiation at contacts between C-nociceptors and spinal-PAG projection neurons.

<p>(A, B) A typical example (A) and quantitative summary (B) of levels of phosphorylated MLC in L4-L5 DRGs of SNS-PKG-I^−/− mice and PKG-I^fl/fl littermates in the naïve state or following formalin injection in the hindpaws. (C) Formalin-induced increase in MLC phosphorylation in L4-L5 DRGs is reduced in wild type mice treated with inhibitors of cGMP synthesis, ODQ (25 mg/kg body weight), and LY83583 (12.5 mg/kg body weight). Mean values from three experiments are depicted below the representative Western blot. (D) Photomicrographic images showing an increase in immunoreactivity for phosphorylated myosin light chains in the spinal dorsal horns in wild-type mice following injection of formalin in the hindpaw. Note increased staining in superficial neuropil as well as cell bodies. Scale bars = 100 µm. (E, F) Time-course (E) and quantitative summary at 30 min (F) after the conditioning stimulus of the blockade of spinal synaptic potentiation by ML-7, an inhibitor of myosin light chain kinase (MLCK) (<i>n</i> = 8 slices/group). (G) MLCK blockade does not significantly affect basal transmission upon dorsal root stimulation (3 mA).</p

Requirement of presynaptic PKG-I for spinal facilitation of nociception induced by the NMDA-NO-cGMP pathway and the natriuretic peptide-cGMP pathway.

<p>(A, B) Facilitation of latency to paw withdrawal (PWL) or tail-flick latency (TFL) following intrathecal application of NMDA (100 fmol), the NO-donor or NOC-12 (17 nmol) in PKG-I^fl/fl mice (black squares), but not in SNS-PKG-I^−/− mice (red circles). (C) mRNA in situ hybridisation showing expression of the catalytic subunit of soluble guanylate cyclise (sGC) in populations of small-diameter neurons (black arrows), large-diameter neurons (black arrowheads), and satellite cells (red arrows) in mouse DRG sections. Scale bar represents 50 µm. (D) Facilitation of latency to paw withdrawal or tail-flick following intrathecal application of a natriuretic peptide cocktail (330 pmol each of ANP, BNP, CNP) in PKG-I^fl/fl mice (black squares), but not in SNS-PKG-I^−/− mice (red circles). All data points represent mean ± S.E.M. * indicates significant statistical difference (<i>p</i><0.05, ANOVA, post hoc Fisher's test) as compared to PKG-I^fl/fl mice, respectively. In all experiments, <i>n</i> = 8–10 mice per genotype or treatment group.</p

Presynaptic PKG-I and molecular mediators of spinal synaptic potentiation are required for behavioural manifestation of nociceptive hypersensitivity.

<p>(A, B) Nociceptive hypersensitivity in the intraplantar formalin test (phase II) is significantly reduced upon intrathecal delivery of an IP₃R blocker (2-APB) or an inhibitor of MLC phosphorylation (ML-7) in wild-type mice (A). SNS-PKG-I^−/− mice show significantly reduced formalin responses than PKG-I^fl/fl littermates (B). (C) Comparison of response frequency to von Frey hairs in SNS-PKG-I^−/− mice (<i>n</i> = 10; red squares) and PKG-I^fl/fl mice (<i>n</i> = 10; black squares) prior to and at 12 h and 24 h following unilateral hindpaw inflammation caused by intraplantar injection of Complete Freund's adjuvant (CFA). Note that leftward deviations in stimulus-response curves in the inflamed state over the basal state are much less pronounced in SNS-PKG-I^−/− mice as compared with their control PKG-I^fl/fl littermates. (D, E) Magnitude and time-course of mechanical hypersensitivity to plantar von Frey hair application (panel D) and thermal hyperalgesia to radiant heat (panel E) following unilateral hindpaw inflammation caused by intraplantar injection of Complete Freund's adjuvant (CFA) to SNS-PKG-I^−/− and PKG-I^fl/fl mice are shown. * <i>p</i><0.05 as compared to PKG-I^fl/fl mice, † <i>p</i><0.05 in PKG-I^fl/fl mice or in SNS-PKG-I^−/− mice as compared to their own basal values, and ANOVA of random measures followed by post hoc Fisher's test. (F) Analysis of excitability of peripheral nociceptors in PKG-I^fl/fl mice and SNS-PKG-I^−/− mice. Electrophysiological recordings from C-mechanoceptors (<i>n</i> = 49) and A-δ-type mechanoceptors (AM, <i>n</i> = 43) in the paw skin-saphenous nerve preparation derived from the paws of PKG-I^fl/fl mice revealed an inflammation-induced increase in the frequency of firing evoked by application of pressure via a nanomotor (expressed in terms of displacement). * indicates significant statistical difference (<i>p</i><0.05, ANOVA, post hoc Fisher's test). Similar recordings in inflamed paw skin derived from SNS-PKG-I^−/− mice showed no significant changes between response properties of C-fibers (<i>n</i> = 43 fibers) and AM-fibers as compared to control SNS-PKG-I^−/− mice (<i>n</i> = 43 fibers).</p

Contribution of PKG-I to synaptic long-term potentiation (LTP) at contacts between C-nociceptors and spinal-PAG projection neurons.

<p>(A) Schematic representation of the experimental approach for dorsal root stimulation and whole-cell patch clamp recordings from spino-PAG projection neurons in lamina I. (B, C) Synaptic LTP was observed following conditioning low-frequency stimulation (2 Hz) of dorsal roots in wild-type mice. LTP was preserved upon spinal blockade of inhibitory neurotransmission (panel B) or upon blockade of PKG-I specifically in the postsynaptic neuron via application of a non-permeant inhibitor in the patch pipette (panel C); <i>n</i> = 11–19 slices each. (D–G) Representative traces (D), time course (E), and quantitative summary of LTP (F, G) evoked by conditioning low-frequency stimulation (2 Hz) of dorsal roots in PKG-I^fl/fl mice, but not in SNS-PKG-I^−/− mice; <i>n</i> = 12 slices per genotype. All data are represented as mean ± S.E.M. * <i>p</i><0.05 as compared to basal and † <i>p</i><0.05 as compared to PKG-I^fl/fl mice, ANOVA followed by post hoc Fisher's test.</p