Reduced inflammatory hyperalgesia with preservation of acute thermal nociception in mice lacking cGMP-dependent protein kinase I

Irmgard Tegeder*†, Domenico Del Turco‡, Achim Schmidtko*, Matthias Sausbier§, Robert Feil¶, Franz Hofmann¶, Thomas Deller‡, Peter Ruth§, and Gerd Geisslinger*

*pharmazentrum frankfurt and ‡Institut fu¨r Klinische Neuroanatomie, Klinikum der Johann Wolfgang Goethe-Universita¨t, 60590 Frankfurt am Main, Germany; §Pharmakologie und Toxikologie, Pharmazeutisches Institut, Universita¨t Tu¨ bingen, 72076 Tu¨bingen, Germany; and ¶Institut fu¨r Pharmakologie und Toxikologie der Technischen Universita¨t, 80802 Munich, Germany

Edited by Joseph A. Beavo, University of Washington School of Medicine, Seattle, WA, and approved December 18, 2003 (received for review July 1, 2003) cGMP-dependent protein kinase I (PKG-I) has been suggested to tion on the PNAS web site.) Hence, the exact role of PKG-I in contribute to the facilitation of nociceptive transmission in the nociception remains elusive. spinal cord presumably by acting as a downstream target of nitric In the present study we used PKG-IϪ/Ϫ mice to clearly assess oxide. However, PKG-I activators caused conflicting effects on the role of PKG-I in nociception. Because these mice have a nociceptive behavior. In the present study we used PKG-I؊/؊ mice defective regulation of smooth muscle contraction with vascular to further assess the role of PKG-I in nociception. PKG-I deficiency and intestinal dysfunctions (17, 18), the overall constitution of Ϫ Ϫ was associated with reduced nociceptive behavior in the formalin homozygous PKG-I / mice deteriorates between 5 and 6 weeks assay and zymosan-induced paw inflammation. However, acute of age (17). We therefore used 3- to 4-week-old animals for thermal nociception in the hot-plate test was unaltered. After nociceptive experiments in the present study. spinal delivery of the PKG inhibitor, Rp-8-Br-cGMPS, nociceptive behavior of PKG-I؉/؉ mice was indistinguishable from that of Materials and Methods PKG-I؊/؊ mice. On the other hand, the PKG activator, 8-Br-cGMP Animals. The generation of the PKG-I null allele and genotyping (250 nmol intrathecally) caused mechanical allodynia only in PKG- was done as described (17). Mice were bred and maintained in -I؉/؉ mice, indicating that the presence of PKG-I was essential for the animal facility of the Institut fu¨r Pharmakologie und Tox this effect. Immunofluorescence studies of the spinal cord revealed ikologie der Technischen Universita¨t Munich. For nociceptive Ϫ/Ϫ ϩ/ϩ additional morphological differences. In the dorsal horn of 3- to testing PKG-I and litter-matched PKG-I mice were 4-week-old PKG-I؊/؊ mice laminae I–III were smaller and contained shipped to the Institut fu¨r Klinische Pharmakologie, Klinikum fewer neurons than controls. Furthermore, the density of sub- der Johann Wolfgang Goethe-Universita¨t Frankfurt. Experi- stance P-positive neurons and fibers was significantly reduced. The ments were performed the day after the arrival. Mice had free access to food and water before and during the experiments. The paucity of substance P in laminae I–III may contribute to the Ϯ ؊/؊ mean SD total body mass of wild-type and knockout mice was reduction of nociception in PKG-I mice and suggests a role of Ϯ Ϯ PKG-I in substance P synthesis. 16.2 2.7 g and 14.2 2.8 g, respectively (12.5% difference). The lean body mass differs by Ϸ8%. All experiments were approved by the local Ethics Committee for Animal Research spinal cord ͉ substance P ͉ nitric oxide ͉ pain and conformed to IASP ethical guidelines.

he second messenger cGMP is formed by activation of Nociceptive Testing. Formalin assay. Fifteen microliters of a 5% Tsoluble and particulate guanylyl cyclases and has several formaldehyde solution was injected into the s.c. space at the targets, including cGMP-dependent protein kinase I (PKG-I) dorsal side of the right hind paw. The time spent licking the and PKG-II, of which PKG-I is expressed in the spinal cord (1, formalin-injected paw was recorded in 5-min intervals up to 45 2). Spinally delivered PKG inhibitors reduce formalin-induced min, starting right after formalin injection. The PKG-I inhibitor nociceptive behavior in rats (3, 4), suggesting that PKG-I plays Rp-8-Br-cGMPS (Biolog Life Sciences Institute, Bremen, Ger- an important role in spinal nociceptive processing. It has been many) was delivered onto the lumbar spinal cord by intrathecal speculated that PKG-I mediates hyperalgesic effects of nitric injection as has been described (19). The drug was dissolved in ϩ ϩ oxide (NO) (5). This idea is supported by the observation that artificial cerebrospinal fluid (141.7 mM Na ͞2.6 mM K ͞0.9 2ϩ͞ 2ϩ͞ Ϫ͞ Ϫ͞ PKG-I inhibition causes a reduction of thermal hyperalgesia mM Mg 1.3 mM Ca 122.7 mM Cl 21.0 mM HCO3 2.5 2Ϫ͞ induced by injection of the NO donor, NOC-12 (6). Endogenous mM HPO4 3.5 mM dextrose, bubbled with 5% CO2 in 95% O2 NO is produced by NO synthases, of which neuronal nitric oxide to adjust the pH to 7.2) and injected in a volume of 5 ␮l. The dose synthase (nNOS) is activated and up-regulated after N-methyl- (50 nmol) was 1͞10th of the dose previously found to reduce D-aspartate receptor stimulation (7–10). NO probably acts as a flinching behavior in rats (4, 15). Drug injection was performed retrograde messenger (11, 12) at nociceptive synapses, i.e., it is in short isoflurane anesthesia 10 min before the injection of released from the postsynaptic neuron, diffuses back to the formalin. presynaptic neuron, and stimulates guanylyl cyclases. The latter Mechanical hyperalgesia in zymosan-induced paw inflammation. Mice step links NO to cGMP production and PKG-I activation. were adapted to the test perspex chamber with a grid bottom for PHARMACOLOGY Because inhibition of NOS activity reduces nociception (13, 14), the release of NO is thought to contribute to the development This paper was submitted directly (Track II) to the PNAS office. of hyperexcitability of nociceptive neurons under certain cir- Abbreviation: MPWT, mechanical paw-withdrawal threshold; PKG, cGMP-dependent pro- cumstances. Under the premise that PKG-I is a mediator of NO tein kinase; LTP, long-term potentiation; nNOS, neuronal nitric oxide synthase. at nociceptive synapses, one would expect that PKG-I activation †To whom correspondence should be sent at the present address: NPRG, Department of also causes hyperalgesia. However, effects of the spinally deliv- Anesthesia, Massachusetts General Hospital and Harvard Medical School, 149 13th Street, ered PKG activator 8-Br-cGMP have been conflicting (15, 16). Room 4309, Charlestown, MA 02129. E-mail: [email protected]. (See Supporting Text, which is published as supporting informa- © 2004 by The National Academy of Sciences of the USA

www.pnas.org͞cgi͞doi͞10.1073͞pnas.0304076101 PNAS ͉ March 2, 2004 ͉ vol. 101 ͉ no. 9 ͉ 3253–3257 Downloaded by guest on September 25, 2021 at least 30 min before baseline testing. Fifteen microliters of a 10 tions, 1 ␮m) were obtained by using a Zeiss LSM 510 confocal mg͞ml zymosan (Sigma) suspension in PBS (0.1 M PBS, pH 7.4) laser-scanning microscope. was then injected into the plantar side of the right hind paw. Mechanical hyperalgesia was assessed before zymosan injection Image Analysis. For quantitative analysis of cell and fiber densi- and then hourly up to7hafterzymosan injection. The threshold ties, midlumbar spinal cord sections (L4͞5) from PKG-IϪ/Ϫ mice to mechanical nociceptive stimuli was assessed by means of a (n ϭ 3) and PKG-Iϩ/ϩ mice (n ϭ 3) were used. Quantification punctuated stimulation by using von Frey hairs of different was performed as described (21), with modifications. Sections strengths (0.008, 0.02, 0.04, 0.07, 0.16, 0.4, 0.6, 1, 1.4, 2, 4, and 6 g; stained for substance P and NeuN and sections stained for nNOS Stoelting). They were placed perpendicularly onto the plantar and NeuN were analyzed (n ϭ 5 per animal per staining). surface of the right or left paw and bent slightly to apply Confocal images were captured by using identical settings for all punctuated pressure. The stimuli were applied at five repetitions sections. Images were analyzed with IMAGEJ 1.31 (http:͞͞ each and at increasing order until the paw was withdrawn and rsb.info.nih.gov͞ij). NeuN labeling of neurons was used to then at decreasing order until paw withdrawal stopped. This identify lamina I–III of the dorsal horn. Cell numbers, mean up-and-down testing was repeated after a short rest. The geo- pixel values, and areas of laminae I–III were determined and metric mean of uppermost (increasing testing) and lowest compared by using Student’s t test (SPSS 11.0). Values of P Ͻ 0.05 (decreasing testing) test results was taken as the mechanical were considered as statistically significant. paw-withdrawal threshold (MPWT). These data were log- transformed, and the percent decrease of the withdrawal thresh- Results old was then calculated in relation to the baseline withdrawal Formalin Assay. PKG-IϪ/Ϫ mice spent significantly less time threshold as: % decrease of MPWT ϭ MPWT࿝baseline Ϫ licking the formalin-injected hind paw than PKG-Iϩ/ϩ mice (Fig. MPWT࿝zymosan͞MPWT࿝baseline⅐100. 1). These differences occurred in the first and second phase of Mechanical allodynia induced by 8-Br-cGMP. After two baseline mea- the formalin assay. Injection of the PKG inhibitor Rp-8-Br- surements 250 nmol of 8-Br-cGMP was injected into the sub- cGMPS significantly reduced the licking behavior in PKG-Iϩ/ϩ arachnoid space of the lumbar spinal cord in 5 ␮l of artificial mice but had no effect in PKG-IϪ/Ϫ mice. The licking behavior cerebrospinal fluid. The dose was 1͞10th of the dose previously in PKG-Iϩ/ϩ mice treated with the PKG inhibitor was equivalent found to cause hyperalgesia in rats (15), i.e., it was equivalent to to that of untreated PKG-IϪ/Ϫ mice, suggesting that the dose of the rat dose on a per kilogram basis. Based on these previous Rp-8-Br-cGMPS was sufficient to completely inhibit PKG-I results, 250 nmol intrathecally can be considered as a high dose. activity in the spinal cord. (See Supporting Text and Fig. 4, which is published as supporting information on the PNAS web site, concerning a low dose of Mechanical Allodynia After Injection of 8-Br-cGMP. 8-Br-cGMP (250 8-Br-cGMP.) The mechanical nociceptive threshold was as- nmol intrathecally) caused mechanical allodynia exclusively in sessed at 5, 7.5, 10, 20, 30, 40, 50, and 60 min after 8-Br-cGMP PKG-Iϩ/ϩ mice (Fig. 2A). In PKG-IϪ/Ϫ mice the MPWT re- injection as described above. Reactions of the right and left paw mained constant after injection of this dose of 8-Br-cGMP. The were identical. The percentage decrease of the MPWT was t test comparing the area under the ‘‘percent decrease of obtained after log transformation as described above. MPWT’’ versus ‘‘time’’ curves (AUCMPWT) revealed significant Acute thermal nociception. A hot-plate test (temperature, 52°C; differences between PKG-IϪ/Ϫ and PKG-Iϩ/ϩ mice (P ϭ 0.007). cut-off latency, 40 s; Hot Plate FMI, Fo¨hr Medical Instruments, Seeheim͞Ober-Beerbach, Germany; time resolution, 0.1 s) was Mechanical Hyperalgesia in Zymosan-Induced Paw Inflammation. At performed to assess acute thermal nociception. The test was baseline, the MPWT did not differ between PKG-IϪ/Ϫ and repeated three times for each mouse with a rest of 15 min PKG-Iϩ/ϩ mice (baseline threshold, 0.26 Ϯ 0.06 g and 0.24 Ϯ in between, and the mean latency was used for statistical 0.06 g in PKG-IϪ/Ϫ and PKG-Iϩ/ϩ mice, respectively; Fig. 2B). comparison. After injection of zymosan into the right hind paw, the MPWT decreased in both groups, indicating inflammatory hyperalgesia. Statistics. To compare the nociceptive behavior between groups In the first2hafterzymosan injection, no difference occurred the total licking time (formalin assay), paw-withdrawal latency between knockout and wild-type mice. However, at Ͼ2h, (hot plate), or the area under the MPWT versus time course hyperalgesia decreased in PKG-IϪ/Ϫ mice, whereas it remained (zymosan and high dose 8-Br-cGMP) was subjected to univariate constant up to the end of the observation period at 7 h in ANOVA in case of more than two groups or Student’s t test in PKG-Iϩ/ϩ mice (Fig. 2B). The statistical comparison of the area case of two groups. After ANOVA testing groups were mutually under the MPWT-versus-time curves from 3 to7hafterzymosan compared with t tests with a Bonferroni ␣-correction for mul- injection revealed significant differences between PKG-IϪ/Ϫ and ϩ ϩ tiple comparisons (␣ at 0.05). We used SPSS 11.0 for statistical PKG-I / mice (P ϭ 0.04). evaluation. Acute Thermal Nociception in the Hot-Plate Test. No difference Immunofluorescence. Mice were intracardially perfused with 0.9% occurred in the paw-withdrawal latency between PKG-IϪ/Ϫ and saline followed by 4% paraformaldehyde in 0.1 M PBS (pH 7.4) PKG-Iϩ/ϩ mice (Fig. 2C; P ϭ 0.88). under deep pentobarbital anesthesia (300 mg͞kg). The spinal cord (lumbar enlargement) was removed and postfixed in the Immunofluorescence Studies. Macroscopically, the spinal cord of same fixative overnight (4°C), and 30-␮m-thick transversal sec- PKG-IϪ/Ϫ mice was thinner than that of PKG-Iϩ/ϩ mice with tions were cut on a vibratome. similar body weight (Ϯ0.5 g). Brightfield micrographs of lumbar Free-floating sections were incubated in blocking buffer (5% sections revealed a reduced ventral-to-dorsal diameter in PKG- normal goat serum͞0.3% Triton X-100 in 0.1 M PBS) for1hat IϪ/Ϫ mice (Fig. 3 A and B). Labeling of neuronal cell bodies with room temperature and incubated overnight at 4°C with primary an antibody against the neuron-specific nuclear protein (NeuN) antibodies directed against PKG-I (1:500; ref. 20), substance P showed the more compact structure of the dorsal horn in (1:500; Santa Cruz Biotechnology), NeuN (1:1,000; Chemicon), PKG-IϪ/Ϫ mice (Fig. 3 C and D). In particular, the superficial N200 (1:1,000; Chemicon), and nNOS (1:500; Alexis Biochemi- laminae I–III were significantly reduced in size (Table 1). The cals, Gru¨nberg, Germany). Binding sites were visualized with changes in size were also observed by immunostaining with the Alexa488- or Alexa568-conjugated species-specific secondary neurofilament antibody N200 (Fig. 3 E and F). In control animals antibodies (Molecular Probes). Confocal images (optical sec- laminae II and III showed only weak axonal labeling and, thus,

3254 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0304076101 Tegeder et al. Downloaded by guest on September 25, 2021 Fig. 1. (A) Time course of the formalin-induced licking behavior in PKG-Iϩ/ϩ (F, n ϭ 8) and PKG-IϪ/Ϫ (E, n ϭ 8) mice. Fifteen microliters of 5% formaldehyde solution was injected s.c. into the right hind paw at time zero, and the time spent licking the injected paw was measured in 5-min intervals for 45 min. Eight mice were used in each group. (B) The bar chart shows the total licking time (0–45 min) after formalin injection in PKG-Iϩ/ϩ and PKG-IϪ/Ϫ mice. Where indicated, 50 nmol of Rp-8-Br-cGMPS (PKG inhibitor) were delivered onto the lumbar spinal cord by injection into the subarachnoid space at the level of L4͞L5 (n ϭ 4 in each group). *, Statistically significant difference, P Ͻ 0.05.

these laminae could readily be recognized as black areas. In Discussion Ϫ Ϫ PKG-I / mice the width of these laminae was clearly smaller We show that cGMP kinase-I deficient mice have reduced ϩ ϩ than in PKG-I / mice. This reduction in size was accompanied formalin-evoked nociception and inflammatory hyperalgesia, by a significant reduction in the number of neurons (Table 1). To whereas reactions to tactile and thermal stimuli to noninflamed study the distribution of functionally important subpopulations paws are normal. This result suggests that the synaptic trans- of neurons located within laminae II and III, substance P- mission of innocuous tactile and heat stimuli is PKG-I- containing neurons and nNOS-positive neurons were also stud- independent, whereas the development and maintenance of ied. Compared with control animals (Fig. 3G), substance P- hyperexcitability of nociceptive neurons involves PKG-I. This immunoreactive neurons and fibers were scarce in superficial finding is in line with previous results obtained with Aplysia. laminae in PKG-IϪ/Ϫ mice (Fig. 3H). Quantitative analysis Noxious stimuli to the mollusc caused long-term hyperexcitabil- ity of nociceptive sensory neurons that required NO͞cGMP͞ revealed a significant reduction of substance P labeling in Ϫ/Ϫ PKG activation and transcription (5). In addition, the late phase PKG-I mice (Table 1). In contrast, no obvious differences in of hippocampal long-term potentiation (late-LTP) is reduced in the distribution of nNOS labeling was found (Fig. 3 I and J and adult PKG-I knockout mice with a hippocampus-specific dis- Table 1). To demonstrate the colocalization of nNOS (Fig. 3I) ϩ ϩ ruption of the PKG-I gene (22). Because LTP is a memory-like and PKG-I (Fig. 3K) in PKG-I / mice, double-immunofluo- mechanism that resembles the activity-dependent ‘‘windup’’ of rescence was used (Fig. 3L). Confocal microscopic imaging nociceptive neurons (11, 12, 23), the function of PKG-I in the revealed numerous double-labeled fibers and cell bodies within sensitization of nociceptive neurons is probably similar to its role the superficial layers (Fig. 3 I, K, and L Insets). in late-LTP. PHARMACOLOGY

Fig. 2. (A) Time course of the percentage change of the MPWT after spinal delivery of the PKG activator 8-Br-cGMP (250 nmol) in PKG-Iϩ/ϩ (F, n ϭ 8) and PKG-IϪ/Ϫ (E, n ϭ 8) mice. The paw-withdrawal threshold was assessed with von Frey hairs. 8-Br-cGMP was injected at time zero. Comparison of the area under the curves revealed a statistically significant difference between PKG-Iϩ/ϩ and PKG-IϪ/Ϫ mice (P Ͻ 0.05). (B) Time course of the percentage change of the MPWT after s.c. injection of zymosan at the plantar side of the right hind paw in PKG-Iϩ/ϩ (F, n ϭ 8) and PKG-IϪ/Ϫ (E, n ϭ 8) mice. The paw-withdrawal threshold was assessed with von Frey hairs. Zymosan was injected at time zero. (C) Paw-withdrawal latency (PWL) as assessed in the hot-plate test (52°C, 40-s cut-off latency) in PKG-Iϩ/ϩ (n ϭ 4) and PKG-IϪ/Ϫ (n ϭ 4) mice. The PWL was measured three times in each animal with a rest of 15 min between measurements.

Tegeder et al. PNAS ͉ March 2, 2004 ͉ vol. 101 ͉ no. 9 ͉ 3255 Downloaded by guest on September 25, 2021 Fig. 3. Brightfield micrographs of lumbar sections of the spinal cord in PKG-Iϩ/ϩ (A) and PKG-IϪ/Ϫ (B) mice. Note the difference in dorsoventral diameter. Dorsal horn of the lumbar spinal cord in PKG-Iϩ/ϩ (C) and PKG-IϪ/Ϫ (D) mice immunostained for NeuN. Note the more compact structure of the dorsal horn in PKG-IϪ/Ϫ mice (arrow) compared with PKG-Iϩ/ϩ mice. Dorsal horn of the lumbar spinal cord in PKG-Iϩ/ϩ (E) and PKG-IϪ/Ϫ (F) mice immunostained for N200. The fiber-poor superficial layers, which contain mostly cell bodies, are broader in PKG-Iϩ/ϩ mice than in PKG-IϪ/Ϫ mice (arrow). Dorsal horn of the lumbar spinal cord in PKG-Iϩ/ϩ (G) and PKG-IϪ/Ϫ (H) mice immunostained for substance P. Note the numerous substance P-positive neurons in the superficial laminae of PKG-Iϩ/ϩ mice (arrow). In contrast, substance P-positive neurons are scarce in these layers in the PKG-IϪ/Ϫ mouse. Dorsal horn of the lumbar spinal cord in PKG-Iϩ/ϩ (I) and PKG-IϪ/Ϫ (J) mice immunostained for nNOS. Although the nNOS-immunoreactive neurons and fibers appear to be more densely packed in the superficial layers, the overall staining pattern of PKG-Iϩ/ϩ and PKG-IϪ/Ϫ mice is similar. (Inset) High-magnification confocal image of nNOS-positive neurons (arrowhead) and fibers. Same section is shown in K and L.(K) Dorsal horn of the lumbar spinal cord in a PKG-Iϩ/ϩ mouse immunostained for PKG-I (same sections as shown in I). PKG-I immunoreactivity is distributed in a pattern that is similar to the one observed for nNOS immunoreactivity. (Inset) High-magnification confocal image of PKG-I-positive neurons (arrowhead) and fibers. (L) Dorsal horn of the lumbar spinal cord in a PKG-Iϩ/ϩ mouse immunostained for nNOS (green) and PKG-I (red). Double-labeled fibers appear yellow. (Inset) High-magnification confocal image of a double-labeled neuron (arrowhead) and numerous double-labeled fibers. (Scale bars: A and B, 200 ␮m; C–L, 100 ␮m; I, K, and L Insets,10␮m.)

PKG-I is thought to act as a downstream mediator of NO. immunofluorescence data of the present study, which show a Inhibition of NO production reduces hyperalgesia, suggesting colocalization of PKG-I and nNOS in spinal cord neurons and that NO facilitates nociceptive transmission (6, 13, 24). PKG axons, provide additional support for the suggested cooperation inhibitors reduced NO-donor or N-methyl-D-aspartate-evoked between NO and PKG-I. hyperalgesia (6), supporting the suggested link between NO and PKG-I additionally regulates the proliferation of sensory PKG-I. PKG inhibitors also blocked hippocampal LTP (25, 26), neurons during embryogenesis (27), and its deficiency caused again indicating similarities between both mechanisms. The axon guidance defects of nociceptive neurons within the devel- oping dorsal root entry zone (2). The present results show that, in terms of formalin-induced nociception, PKG-I inhibition is as Table 1. Quantitative analysis of immunofluorescence studies for effective as PKG-I deficiency. This finding suggests that the laminae I–III previously observed axon guidance defects of nociceptive neu- Ϫ/Ϫ Lamina I–III area, NeuN IR rons in PKG-I mice do not essentially contribute to the Ϫ/Ϫ ␮m2 neurons SP (IF) nNOS (IF) observed alterations of nociception in 3- to 4-week-old PKG-I mice in this test. The defects in axon growth and connectivity and Ϫ͞Ϫ PKG-I 107,332.4 Ϯ 5,441.4* 223.1 Ϯ 19.8* 52.3 Ϯ 2.5* 67.1 Ϯ 9.2 the resulting paucity of fibers in the dorsal funiculus (2) also ϩ͞ϩ PKG-I 133,282.3 Ϯ 5,922.6 289.3 Ϯ 28.8 65.0 Ϯ 4.4 73.0 Ϯ 3.3 apparently do not impair acute heat or mechanical nociception, Data represent the mean Ϯ 2 SEM. SP, substance P; nNOS, neuronal nitric because paw-withdrawal latency in the hot-plate test and base- Ϫ/Ϫ ϩ/ϩ oxide synthase; IF, immunofluorescence intensity of SP- or nNOS-positive line von Frey responses were equal in PKG-I and PKG-I structures (neurons and fibers). *, P Ͻ 0.05, statistically significant difference. mice. However, the defects during embryogenesis may explain

3256 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0304076101 Tegeder et al. Downloaded by guest on September 25, 2021 the observed size differences of the spinal cord with a remark- appears to be involved in PKG effects on LTP, because the able reduction of the dorsoventral diameter in PKG-IϪ/Ϫ mice. translation inhibitor anisomycin abolished differences in late Although PKG-IϪ/Ϫ mice were somewhat smaller and, in gen- hippocampal LTP between control and hippocampus-specific eral, have less adipose tissue than their littermates, it seems PKG-I knockout mice (22). It is therefore conceivable that unlikely that this dorsoventral size reduction is completely PKG-I regulates the expression of substance P precursors what accounted for by leaner bodies. would explain the reduced number of substance P-immunore- Ϫ/Ϫ We found that the density of substance P-immunoreactive active neurons and fibers in PKG-I mice. The paucity of Ϫ/Ϫ substance P in laminae I–III may contribute to the reduced neurons and fibers was significantly reduced in PKG-I mice. Ϫ/Ϫ The potential link between PKG-I and substance P or its nociceptive response in PKG-I mice. In summary, our results suggest that spinal PKG-I is involved precursors has not been directly addressed until now. However, in the facilitation of synaptic transmission of nociceptive stimuli the NO-donor sodium nitroprusside evokes substance P release in the spinal cord in an ongoing activation, whereas acute from spinal cord slices (28). Guanylyl cyclase inhibition prevents heat-induced nociception does not require PKG-I activity. this NO-evoked substance P release, suggesting that this effect is mediated by cGMP (28) and therefore possibly by PKG. PKG This study was supported by Deutsche Forschungsgemeinschaft [Sonder- primarily regulates target protein activity by phosphorylation forschungsbereich 553 (C6), 269 (B7), 391, and Research Fellowship (29) but may also regulate protein expression (30). The latter DFG TE 322࿝2-1].

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