Results of the quantitative analysis of GABA immunoreactivity.

<p>Mean values are shown with ranges in brackets (n = 4).</p

Distribution of sst_2A immunoreactivity in the mouse dorsal horn.

<p><b>a</b>: There is a dense band of immunostaining in laminae I and II, with much lower levels elsewhere in the grey matter. Immunoreactive cells (some marked with arrows) are scattered throughout laminae I and II, and present at much lower density in deeper laminae. The solid line shows the grey-white matter boundary and the dashed line the border between laminae II and III. <b>b–f</b>: The relationship between sst_2A (green), PKCγ (magenta) and the appearance of the dorsal horn with dark-field illumination (DF). Note that although scattered PKCγ cells are present in lamina III, the ventral border of the PKCγ plexus (arrowheads) is at the same level as the ventral edge of sst_2A staining, and that this corresponds to the lamina II/III border as seen with dark-field microscopy. All confocal images are from single optical sections. Scale bar  = 100 µm.</p

NK1r and sst2A in superficial dorsal horn.

<p>Confocal image showing a single optical section through part of laminae I and IIo, scanned to reveal NK1r (green) and sst_2A (magenta). <b>a</b>: Two NK1r-immunoreactive cell bodies (arrows) are visible in lamina I, surrounded by a plexus of labelled dendrites. <b>b</b>: Several sst_2A-immunoreactive cells are visible in the same field (two shown with arrowheads). <b>c</b>: The merged image shows that none of these cells is labelled with both antibodies. Scale bar  = 50 µm.</p

GABA and glycine immunoreactivity in a 0.5 µm thick transverse section of the mouse dorsal horn.

<p><b>a</b> shows the whole mediolateral extent of the dorsal horn stained for GABA, while <b>b</b> and <b>c</b> show the medial part of the dorsal horn at higher magnification, in the same section and in a section reacted for glycine, respectively. Many GABA-immunoreactive cell bodies are scattered throughout the dorsal horn, and although these vary in intensity, they are clearly darker than the immunonegative cells, some of which are marked with asterisks in <b>b</b>. The dark staining in the neuropil represents GABAergic axons and dendrites. Dashed lines show the ventral borders of laminae I, II and III. <b>b</b> and <b>c</b> are serial semithin sections, so the same cells are visible. A few cells with relatively strong glycine immunoreactivity are visible (some marked with arrows) and these are also GABA-immunoreactive. In addition, there are cells that are GABA- but not glycine-immunoreactive (two marked with arrowheads). Scale bars  = 100 µm.</p

Proportions of neurons in laminae I-III that were sst_2A- or NK1r-immunoreactive and numbers of neurons in each lamina.

<p>Mean values are shown with ranges in brackets (n = 4). The estimated total number of neurons in L4 is based on the average length of the segment (1.45 mm) in these 4 mice.</p

Primary antibodies used in this study.

*<p>dilutions used for post-embedding method</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

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

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