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The Journal of Neuroscience, May 15, 1996, W(10):3130-3138 cGMP-Dependent in Dorsal Root Ganglion: Relationship with Synthase and Nociceptive

Yifang Qian,l Daniel S. Chao,’ Daniel R. Santillano,l Trudy L. Cornwell, Angus C. Nairn,4 Paul Greengard,d Thomas M. Lincoln,3 and David S. BredV* 1Deparfment of Physiology and *Program in Biomedical Sciences, University of California San Francisco, San Francisco, California 94 143, 3Deparlmen t of Pathology, University of Alabama at Birmingham, Birmingham, Alabama 35294, and 4DepaHment of Molecular and Cellular Neuroscience, The Rockefeller University, New York, New York IO02 1

Nitric oxide and cGMP influence plasticity of nociceptive pro- and roof plates. Neuronat nitric oxide synthase (nNOS) is co- cessing in spinal cord. However, effecters for cGMP have not expressed with cGKI in sensory neurons during embryonic been identified in sensory pathways. We now demonstrate that development and after peripheral nerve axotomy. The primary cGMP-dependent protein kinase I (cGKI) occurs in the DRGs at target for cGKl in cerebellum, G-substrate, is not present in levels comparable to that in cerebellum, the richest source of developing, mature, or regenerating sensory neurons, indicat- cGKI in the body. Immunohistochemical studies reveal that ing that other serve as effecters for cGKI in sensory cGKI is concentrated in a subpopulation of small- and medium- processing. These data establish sensory neurons as a primary diameter DRG neurons that partially overlap with substance P locus for cGMP actions during development and suggest a role and calcitonin gene-related polypeptide containing cells. Dur- for cGKI in plasticity of nociception. ing development, cGKl expression throughout the embryo is Key words: nociceptive; dorsal root ganglion; nitric oxide; essentially restricted to sensory neurons and to the spinal floor development; substance P; cGMP

Nitric oxide (NO) serves as a cellular mediator for diverse devel- sensory processing derives from pharmacological studies. NOS opmental and physiological processes (Marletta, 1993; Moncada antagonists do not alter baseline responses indicating that NO and Higgs, 1993; Bredt and Snyder, 1994a; Nathan and Xie, 1994; does not function as a fast transmitter at primary afferent syn- Garthwaite and Boulton, 1995). Neuronal type NO synthase apses. NOS antagonists, however, do prevent spinal sensitization (nNOS, or type I) is localized to a discrete population of neurons of nociceptive reflexes (Moore et al., 1991; Kitto et al., 1992; in the embryonic and mature (Bredt et al., 1991; Meller et al., 1992; Meller and Gebhart, 1993). This NO- Bredt and Snyder, 1994b). nNOS is a /- dependent sensitization contributes to hyperalgesia after periph- dependent that is stimulated by activation of NMDA type eral injury and is also blocked by NMDA receptor antagonists glutamate receptors (Garthwaite et al., 1988; Knowles et al., 1989; (Dickenson, 1990). Bredt and Snyder, 1990). NO formed during excitatory transmis- NO signaling occurs primarily through activation of soluble sion participates in synaptic plasticity mediated by NMDA recep- isoforms of guanylyl cyclase. In the vascular and nervous systems, tor activity (Schuman and Madison, 1994). cGMP-dependent protein (cGKs) serve as major effecters A role for NO in sensory signaling was initially suggested based for NO and cGMP (Lincoln et al., 1994). Endothelial-derived NO on the specific localization of nNOS in sensory pathways (Bredt et stimulates guanylyl cyclase and subsequently cGK in vascular al., 1990; Aimi et al., 1991). In normal adult mammals, nNOS is smooth muscle leading to relaxation (Ignarro, 1993; expressed in a few small- and medium-diameter DRG neurons, Moncada and Higgs, 1993; Warner et al., 1994). Similarly, phys- and in most of these cells, nNOS coexists with calcitonin gene- iological actions of NO in sensory pathways appear to involve related peptide (CGRP) and rarely with substance P @hang et al., cGMP (Meller and Gebhart, 1993). NO-mediated hyperalgesia is 1993). By contrast a large percentage of DRG neurons contain blocked by extracellular hemoglobin (Kitto et al., 1992), indicating nNOS transiently during embryonic development (Bredt and Sny- a role for intercellular NO signaling. Furthermore, membrane der, 1994b) and after peripheral nerve damage @hang et al., permeable cGMP analogs mimic the effects of NO on sensory 1993). Direct evidence for involvement of NO in plasticity of responsiveness (McGehee et al., 1992; Niedbala et al., -_--l--“-.--.------~.- . .“_-_~-.-...- 1995), Downstream effecters for cGMP, however, have not pre- Received Oct. 5, 1995; revised Feb. 12, 1996; accepted Feb. 22, 1996. viously been characterized in sensory neurons. This work was supported by grants (to D.S.B.) from the NationaI Science Foun- Molecular studies have identified two genetic loci encoding dation, the Lucille P. Markey Charitable Trust, and the National Association for Research on Schizophrenia and Depression, the McKnight Endowment Fund for mammalian cGKs. The first gene product characterized, cGKI, Neuroscience, the Esther A. and Joseph Klingenstein Fund, and National Institutes was purified and sequenced from soluble extracts of vascular of Health Grants HL 34646 (T.M.L.) and MH-40899 (P.G.). We thank Allan smooth muscle from bovine lung (Lincoln et al., 1977; Flockerzi et Basbaum for helpful discussions and for review of this manuscript. Correspondence should be addressed to David S. Bredt, Department of Physiol- al., 1978; Takio et al., 1984). Molecular cloning of the cDNA ogy, School of Medicine, University of California at San Francisco, 513 Parnassus demonstrated N-terminal alternative splicing yielding cGKIa Avenue, San Francisco, CA 94143-0444. Dr. Qian’s present address: Department of Psychiatry, San Mateo County General and cGKI/3 isoforms (Wernet et al., 1989). More recently, a Hospital, San Mateo, CA 94113. membrane-associated cGKI1 has been cloned from brain (Uhler, Copyright 0 19% Society for Neuroscience 0270-6474/96/i 63 130-09 $0500/O 1993) and from the epithelium of intestinal mucosa (Jarchau et Qian et al. l cGMP-Dependent Protein Kinase in Dorsal Root Ganglion J. Neurosci., May 15, 1996, 76(10):3130-3138 3331

al., 1994). In the CNS cGK enzyme activity is selectively enriched Cb Cx DRG in the cerebellum where high levels of cGKI protein are histo- A chemically apparent in Purkinje neurons (Lohmann et al., 1981). 200- Low levels of cGK activity found in forebrain tissues appear to primarily represent cGKI from vascular smooth muscle (Loh- 97- mann et al., 1981). A prominent target for cGK, G-substrate, is - - exclusively found in cerebellum (Detre et al., 1984). The prominent functions for NO and cGMP in sensory plastic- 68- ity led us to investigate the possible expression of cGKs in sensory neurons. Strikingly, we demonstrate that cGKI occurs in DRG at levels equivalent to that in cerebellum. cGKI is found in small- 43- and medium-diameter neurons of DRG and is concentrated in axonal processes in laminae I and II of spinal cord, consistent with a-cGK1 known roles for NO and cGMP in nociceptive processing. During B embryogenesis, cGKI is found only in DRG neurons and cells of 200- the spinal floor and roof plates. Whereas cerebellar Purkinje -- soma contain both cGKI and G-substrate, sensory neurons lack 97- G-substrate, suggesting that novel targets mediate actions of cGKI in sensory neuron development and plasticity. 68- MATERIALS AND METHODS Antisera. The following primary polyclonal rabbit antibodies were used: cGKI (Lohmann et al., 1981; Wyatt et al., 1991), nNOS (Bredt et al., 1990), substance P (Peninsula Laboratories), CGRP (Peninsula Labora- 43- tories). For double-immunofluorescence labeling, a polyclonal guinea pig antiserum to substance P was used (Too and Maggio, 1991). a-nNOS Ajinity purification of G-substrate antibody. Polyclonal antibody to G-substrate (Dolphin et al., 1983) was further affinity purified by presorb- ing nonspecific antibodies with a crude cerebral cortical extract. Rat cerebral cortex was homogenized in 10 volumes of 25 mM Tris-HCl buffer, pH 7.4, 1 mM EDTA, 100 PM PMSF and diluted to 5 mg protein/ml. Ten micrograms of antibody to G-substrate (Dolphin et al., 1983) were incubated overnight with 5 mg of cortical extract. Soluble antibodies were recovered by filtration through a 0.44 pm mesh. Western blotting. Tissues were dissected and homogenized in 10 vol- umes of Tris-HCl buffer, pH 7.4, containing 1 mM EDTA and 100 PM PMSF. Acid soluble extracts were prepared using the method of Aswad and Greengard (1981). Proteins were separated by SDS-PAGE and transferred to PVDF membranes, and blots were incubated in blocking solution containing 3% bovine serum albumin in PBS and 0.1% Tween 20. Antibodies were diluted in this same buffer and hybridized to blots overnight. Immunoreactive bands were visualized by ECL according to the manufacturer’s specifications (Arnersham, Arlington Heights, IL). Immunohistochemistry. Adult and embryonic rats were anesthetized a-G sub with pentobarbital and perfused with 4% freshly depolymerized parafor- maldehyde in 0.1 M phosphate buffer. Tissues were removed and postfixed Figure 1. cGMP-dependent protein kinase but not nNOS or G-substrate in paraformaldehyde for 1 hr at 4°C. Tissues were cryoprotected over- is enriched in dorsal root ganglion extracts. A, Western blotting shows that night in 20% sucrose. Thin sections were cut on a cryostat (6 pm), and cGKI occurs at similar levels in crude extracts of DRGs as in cerebellum free-floating sections (40 pm) were cut on a sliding microtome. Endog- (Cb) but is only weakly detectable in cerebracortical (Cx) tissue. B, nNOS enous peroxidase activity was inactivated by incubating sections in 0.5% is most enriched in cerebellum, is moderately expressed in cerebral cortex, H,O, for 15 min. Sections were blocked for 1 hr in PBS containing 1.5% and is only faintly detectable in DRG. C, G-substrate is only detectable in NGS and then incubated overnight in the same buffer containing diluted cerebellar extracts and does not occur in cortex or DRG. A, B, Crude antiserum. Immunohistochemical staining used an avidin/biotin/peroxi- tissues were homogenized in 25 mM Tris-HCl buffer, pH 7.4, containing 1 dase system (ABC Elite; Vector Laboratories, Burlingame, CA) accord- mM EDTA, 100 WM PMSF, and 0.5% Triton X-100. Soluble proteins were ing to the manufacturer’s instructions. Peroxidase staining was developed resolved by 10% SDS-PAGE (80 pg/lane). For C, acid-soluble extracts using 3,3’-DAB as the chromogen. For indirect immunofluorescence, were separated on a 15% SDS-PAGE gel (50 pg/lane). Immunoreactive secondary goat anti-rabbit Cy-3, and donkey anti-guinea pig FITC con- bands were detected by ECL. Positions of molecular weight standards (in jugated antibodies were used according to the manufacturer’s specifica- kDa) are indicated on the left. tions (Jackson Laboratories, 1:200). Surgery. Lesions of the rat sciatic nerve were performed by exposing the right nerve in the mid thigh and crushing it for 10 set with a number 6 jeweler’s forceps. After the lesion, the skin was sutured and the animals were housed in isolation for 18 d. Affected DRGs were isolated by tracing DRG, cerebellum, and cerebral cortex of an adult rat were re- the sciatic nerve back to the spinal cord (LA or L5). Isolated DRGs were solved by SDS-PAGE and probed with antisera to cGKI and dissected, fixed in paraformaldehyde, and stained as described above. nNOS (Fig. L4,B). Each antiserumreacts specifically with a single band of appropriate molecular weight. cGKI (MW - 74,000) RESULTS occursat comparablelevels in DRG extracts as in cerebellum,the cGKl is expressed at high levels in DRG extracts richest known sourceof cGKI in the body (Fig. L4). As previously We first characterized the specificity of antisera to cGKI and reported (Lohmann et al., 1981), cGKI is present at very low nNOS by Western blotting. Crude protein extracts from lumbar levels in forebrain extracts. nNOS (MW - 160,000)occurs most 3132 J. Neurosci., May 15, 1996, 76(10):3130-3138 Qian et al. l cGMP-Dependent Protein Kinase in Dorsal Root Ganglion

Figure 2. cGKI is enriched in small- and medium-diameter DRG neurons, and ex- pression does not change after axotomy. A, cGKI is heterogeneously expressed in spe- cific neurons of the rat L5 DRG. Examples of densely stained small- and medium- diameter neurons are indicated with a solid arrow. Unstained large cells are identified by an open arrow (magnification 200X). B, C, Expression of cGKI does not dramatically change 18 d after unilateral sciatic nerve axotomy as a similar pattern and density of staining of DRG cells is noted contralateral (CIV) and ipsilateral (ZP) to the lesion (100X). D, E, nNOS expression is dramati- cally induced in ipsilateral L5 DRG neurons 18 d after sciatic nerve axotomy (100X). Ten micrometer cryostat sections of DRG were processed for cGKI or nNOS immunohisto- chemistry and photographed with light-field microscopy. prominently in cerebellar extracts and is only faintly detectable in nNOS, the upstreamactivator cGKI, is present in only l-2% of mature DRG tissue (Fig. 1B). To ensurespecificity of immuno- rat lumbar DRG neurons (Aimi et al., 1991). After peripheral histochemicalstaining (seebelow) we used two different affinity nerve axotomy, nNOS expressionis induced in specific DRG purified antisera to cGKI. Both of these antisera were raised neurons, that correspond to small- and medium-diameter cells againstintact purified cGKI protein and have been well charac- (Zhang et al., 1993). To determine the relationship between terized in previousimmunohistochemical studies (Lohmann et al., nNOS and cGKI, we performed unilateral sciatic nerve axotomy 1981; Wyatt et al., 1991). Both antiseragive essentiallyidentical and evaluated cGKI and nNOS labeling of DRG cells ipsilateral patterns of staining in all tissuesexamined. For consistency,all and contralateral to the lesion. We confirmed dramatic induction cGKI histochemistry presented here used the affinity purified of nNOS after axotomy (Fig. 2QE) but found no significant antiserumdescribed by Wyatt et al. (1991). Immunoreactivity was changein cGKI stainingup to 18 d after the lesion (Figs. 2&C). not observed in sectionslabeled with nonimmune serum or in Central projections of primary nociceptors terminate in lami- incubations lacking primary antibody (data not shown). nae I and II of the spinal cord. Immunohistochemicalstaining of L5 spinal cord segmentsdemonstrated cGKI in nerve terminalsof cGKl occurs in small- and medium-diameter sensory neurons lamina I and II (Fig. 4). Spinal neurons and white matter tracks are devoid of cGKI staining. The distribution of cGKI in dorsal Immunohistochemicalanalysis reveals that cGKI is discretely spinalcord is similarto that of substanceP (Fig. 4) (Chung et al., localized to specific neurons in lumbar DRG (Fig. U). cGKI 1988). nNOS containing fibers, by contrast, are found in all layers staining of DRG cell bodies is heterogeneous;some cells are of the spinal cord, but are most concentrated in the lamina II, intensely stained, others stain weakly and somenot at all. DRG confirming previous studies(Fig. 4) (Bredt et al., 1991; Zhang et neuronsintensely stained by cGKI were all of small (<32 pm) or al., 1993). There are also many nNOS containing small interneu- medium (32-50 pm) diameter, the classof neurons involved in rons that are most numerousin lamina II. nociceptive processing.Weakly stained neurons were of both mediumand large (~50 pm) cell diameters.Because many small- cGKl is concentrated in embryonic sensory neurons and medium-diametersensory neurons contain substanceP or and spinal floor plate CGRP, neuropeptidesthat participate in nociceptive transmission Although present in only few mature DRG neurons, nNOS is (Hokfelt, 1991) we stained adjacent thin (6 pm) sections for transiently expressedin many sensoryneurons during embryonic cGKI and substanceP or CGRP. We found that neuronsintensely development(Bredt and Snyder, 1994b). We therefore evaluated stainedfor cGKI partially overlap with neuronscontaining sub- expressionof cGKI during development. Western blotting indi- stanceP (Fig. 3&l’) or CGRP (Fig. 3&B’). We also conducted cated that cGKI is present in extracts of DRG from embryonic double-immunofluorescentlabeling studies of L5 DRG using day 15 (E15) rat, but that cGKI is barely detectable in brain antibodiesto cGKI and substanceP (Fig. 3C,C’). We found that extracts at this stage (Fig. 5). nNOS is found at similar levels in cGIU immunoreactivity was found in essentiallyall substanceP El5 DRG and brain extracts. Immunohistochemistryof rat em- positive cells, whereas substanceP occurred in 641356cGIU bryos demonstratedthat cGKI is restricted to developing sensory containing neurons. pathways (Fig. 6). In sagittal sectionsof El5 embryos, cGKI is Qian et al. l cGMP-Dependent Protern Kinase in Dorsal Root Ganglion J. Neurosci., May 15, 1996, 76(10):3130-3138 3133

Figure 3. cGKI partially overlaps with substance P and CGRP in DRG neurons. Staining of adjacent 6 pm sections shows that cGKI (A) occurs in a greater fraction of DRG cells than substance P (A’). In certain cells, cGKI coexists with substance P (solid avow), whereas others contain cGKI but not substance P (open arrow) (200X). A cell labeled for cGKI and CGRP (solid arrow) is shown in the middle panels (B, B’). These panels also show a medium-diameter neuron stained for cGKI but not CGRP (open arrow) (400X). Double-immunofluorescent labeling shows colocalization of cGKI (C) and substance P (C’) in a single neuron.

concentrated in cell bodiesof the DRG, aswell as fine processes neurons(Figs. 7C-E). Immunostainingoccurs both in cell bodies in dorsal spinal cord and periphery. cGKI is not apparent in any and in central and peripheral axonsof embryonicsensory neurons. other cell type or tissuein El5 embryos.Micrographs of horizon- cGKI labeling of sensoryprojections to the dorsal spinal cord tal sectionsrevealed that cGKI occurs in sensoryganglia as early (Figs. 7A,C,E) accountsfor stainingseen in dorsal cord (Fig. 6). as El2 (Fig. 7A). Interestingly, cGKI is also prominently ex- cGK1 also labelscutaneous branches of sensoryaxons throughout pressedin the spinalfloor and roof plate at El2 (Fig. 7B). Spinal the embryo (Fig. 7G), which accounts for staining of fine pro- neuronsthemselves do not expresscGKI in embryonic animals cessesin the periphery seenat low magnification (Fig. 6). (Figs. 7&E). By El5 labeling of the floor plate is nearly absent, To evaluate possibleregulation of cGKI by NO during devel- whereascGKI stainingremains prominent in a subsetof sensory opment, we stainedappropriate sectionswith antibodiesto nNOS.

Figure 4. cGKI is colocalized with sub- stance P in dorsal spinal cord. A, cGKI is restricted to neuronal fibers in laminae I and II of the spinal cord. cGKI is not present in white matter tracks of the dor- sal columns (DC) or in spinal neuron cell bodies (50X). B, cGKI also stains the vascular smooth muscle of blood vessels (BV, 100X). C, nNOS occurs in neuronal processes and throughout the dorsal horn (OH) and is present in occasional spinal interneurons (100X). D, Sub- stance P is colocalized with cGKI in lam- inae I and II of the dorsal horn (100X). 3134 J. Neurosci., May 15, 1996, 76(10):3130-3138 Qian et al. l cGMP-Dependent Protein Kinase in Dorsal Root Ganglion

DRG cells as well as the central and peripheral projections of El5 thesesensory neurons (Fig. 7F,H). As previously reported (Bredt A Cb Br DRG and Snyder, 1994b), nNOS is also present in embryonic spinal neurons of the intermediolateral cell column that lack cGKI 2(H)- (Fig. 7E,F). G-substrate is enriched in Purkinje cell soma but not in sensory neurons Purkinje neuronsof the cerebellum,the only other neuronal cell type to expresscGKI at high density (Lohmann et al., 1981), are also enriched with G-substrate (Detre et al., 1984), a potent protein target for cGKI. This localization was basedon studies 68- showingdecreased G-substrate levels in the cerebellumof mutant mice lacking Purkinje cells (Detre et al., 1984). To directly eval- uate the distribution of G-substratein cerebellum and DRG, we generated an affinity purified antiserumby taking advantageof the absenceof G-substratein cerebral cortex (Detre et al., 1984). A G-substrateantiserum (Nairn et al., 1982; Dolphin et al., 1983) was preabsorbedwith crude protein extracts from rat cerebral cortex to remove cross-reactingantibodies. Western blotting of a-cGKI cerebellar extracts indicates that this affinity purified antiserum reactswith a major 23,000Da band, correspondingto G-substrate (Detre et al., 1984). By contrast, no bands were detected in extracts of cerebral cortex or DRG (Fig. 1C). We also failed to detect G-substratein brain or DRG extracts from embryonic rat (data not shown). Immunohistochemicalstudies demonstrate that G-substrateis most concentrated in Purkinje neuronsof the cerebellum(Fig. 8). Neuronal fibers in thalamusand pyramidal layer of the hippocam- 97- pus also contain G-substrateimmunoreactivity. cGKI is concen- trated together with G-substratein Purkinje cells of the cerebel- lum aspreviously reported (Fig. 8; Lohmann et al., 1981).cGIU is also enriched in pyramidal cells and dentate granule cells of the hippocampus.By comparison, nNOS displays a more general neuronal distribution, as describedpreviously (Bredt et al., 1990). High power micrographs of the cerebellum demonstrate that 68. G-substrateis specifically concentrated in Purkinje cell soma(Fig. 9). By contrast, cGKI is present throughout the Purkinje cell, in the apical dendrites, cell soma and axon as reported previously a-nNOS (Lohmann et al., 1981). Specific G-substrateor cGKI immunore- activity is not apparent in cells in the granular layer or molecular Figure 5. cGK1 is uniquely enriched in DRG tissue extracts during layers of cerebellum. nNOS in cerebellum occurs in a comple- embryonic development. A, Western blotting shows that cGK1 is mentary distribution, in basket cells of the molecular layer and present in DRG, but not brain tissue, in El.5 rat extracts. cGK1 from neurons as reported previously (Bredt et al., 1990). El5 DRG comigrates with a similar 74 kDa band from adult cerebel- lum (Cb). B, Probing a duplicate blot shows that nNOS is expressed at comnarable levels in El5 brain and DRG extracts. Solubilized tissue DISCUSSION extracts were separated by 10% SDS-PAGE. Five times more protein was loaded from embryonic tissues (100 &lane) than from cerebellum A principle finding of this work is that a subsetof sensoryneurons (20 &lane). Note that El5 DRG extracts were unavoidably contam- is dramatically enriched in cGKI. Previous biochemicaland his- inated with adjacent spinal tissues and may therefore underestimate tochemical studiessuggested that Purkinje neurons of the cere- true enrichment of cGK1. bellum were the only cells to be enriched with cGIU at levels comparableto those in vascularsmooth muscle(Lohmann et al., 1981).Much lower levels of cGKI have been detected in other cell Low-power dark field imagesshow that nNOS occurs together types including macrophages,neutrophils, osteoclasts,and vascu- with cGKI in El5 DRG cells and sensoryprojections (Fig. 6). As lar endothelial cells (for review, see Lincoln, 1995). In these previously reported, nNOS also occursin other embryonicsensory tissuescGKI is associatedwith cytoskeletal proteins such as vi- cells suchas primary olfactory neurons(Bredt and Snyder, 1994b; mentin (MacMillan-Crow and Lincoln, 1994). Similarly, we have Roskamset al., 1994),which lack cGKI (Fig. 6). nNOS immuno- recently noted that cGKI is present at low levels in skeletalmuscle reactivity is alsoapparent in cerebral cortical plate, neuronsof the cells and is selectively associatedwith neuromuscularendplates midbrain, myenteric neurons,epithelial cells of the gastrointesti- (D. Chao, F. Silvagnano,T. Cornwell, T. Lincoln, and D. Bredt, nal tract, and certain epithelial cells of the embryonickidney all of unpublishedobservations). which lack cGKI (Fig. 6) (Bredt and Snyder, 199413).High-power The abundanceof cGKI in small- and medium-diameterneu- micrographsshow that nNOS is colocalized with cGKI in El5 rons of the DRG has important implications for mechanismsof Qian et al. l cGMP-Dependent Protein Kinase in Dorsal Root Ganglion J. Neurosci., May 15, 1996, 16(10):3130-3138 3135

Figure 6. cGKI and nNOS are colocal- ized in embryonic sensory neurons. At E15, cGKI is apparent only in cells and axonal processes of sensory neurons. In- tense staining of the DRG, dorsal cord (DC), and peripheral cutaneous axons (Ax) is noted. nNOS is present in DRG but also occurs in olfactory epithelium (O), cerebrocortical plate (CP), kidney @), adrenalgland iA), and ‘intestine (In). all of which lack cGKI. Twentv mi- \ II 3 crometer cryostat sections of El5 rat em- bryos were stained with antisera to cGKI or nNOS. In these dark-field images, positive staining appears white. Note that fixation conditions for cGKI and nNOS immunohistochemistry are somewhat different and do not allow staining of adjacent sections from the same embryo.

NO-mediated nociceptive processing. In several animal models signalingby NO may participate in spinal sensitization. In this NOS inhibitors have been found to block hyperalgesia subsequent model, neuron-derived NO from the spinal cord would serve asa to peripheral tissue injury (Moore et al., 1991; Niedbala et al., retrograde messengerto increasecGMP levels and activate cGKI 1995). This sensitization is mediated by NMDA receptors and in primary nociceptors.In support of this, high densitiesof nNOS occurs at the level of the spinal cord. In support of this conclusion, are found in neuronal fibers and interneuronsin superficiallayers intrathecal administration of NOS inhibitors or NMDA receptor of the dorsal spinal cord (Fig. 4; Bredt et al., 1991; Zhang et al., antagonists prevents hyperalgesia produced by a variety of exper- 1993). imental stimuli without affecting normal reflex function (Dicken- Presynaptictargets for cGKI that participate in synaptic plas- son, 1990; Meller and Gebhart, 1993). Furthermore, intrathecal ticity are uncertain. In vitro phosphorylation studies identified administration of either an NO donor or NMDA itself produces a G-substrateas a prominent target for cGKI in cerebellum(Detre marked hyperalgesia in nociceptive tests (Aanonsen and Wilcox, et al., 1984). G-substrateis a small heat-stableprotein that shares 1987; Coderre and Yashpal, 1994). These hyperalgesic actions of biophysicalproperties with certain protein phosphataseinhibitors, NO and NMDA appear to be mediated by activation of guanylyl such as DARPP-32 and inhibitor 1. Our immunohistochemical cyclase as they are mimicked by membrane permeant cGMP studiesdemonstrate that G-substrateis restricted to cell somaof analogs and are blocked by guanylyl cyclase antagonists (McGe- cerebellar Purkinje cells and is entirely absent from sensoryneu- hee et al., 1992; Niedbala et al., 1995). rons. This restricted distribution makes G-substrate an unlikely Consistent with our proposed role for cGKI in spinal sensitiza- mediator of synapticplasticity. On the other hand, recent studies tion, pharmacological studies suggest that cGKI mediates synaptic demonstrate that rabphilin-3A is an efficient substratefor phos- plasticity in several other systems. In Purkinje neurons of the phorylation by endogenouscGK in vitro (Fykse et al., 1995). cerebellum, cGKI activity is required for expression of long term Rabphilin3A is a Ca-binding synaptic vesicle protein that is depression, a model of motor learning (Shibuki and Okada, 1991; implicated in exocytosis or vesicular transport (Shirataki et al., but see Linden and Connor, 1995). Similarly, effects of NO and 1993). It will now be important to determinewhether rabphilin 3A cGMP on hippocampal long term potentiation are believed to be or other synaptic vesicleproteins serve as the physiologicalsub- mediated by cGK (Zhuo et al., 1994). Both of these models stratesfor cGKI that mediate presynapticplasticity. involve a Hebbian mechanism (Hebb, 1949); that is, coincident Our developmental studiesyield insight into mechanismsfor activity of the pre- and postsynaptic neuron yields long lasting NO- and cGMP-mediated actionsduring embryonicdevelopment. changes in synaptic efficacy. NO functions as an anterograde nNOS is transiently expressed in developing sensory neurons messenger in cerebellar long term depression, transmitting infor- (Bredt and Snyder, 1994b), and NO has been implicated in neu- mation from granule cell axons to Purkinje cell dendrites (Lev- ronal differentiation (Peunova and Enikolopov, 1995) and axon Ram et al., 1995). By contrast, a retrograde function for NO in outgrowth (Hess et al., 1993). High levels of cGKI expression hippocampal plasticity is suggested by experiments showing that restricted to sensoryneurons during embryonic developmentsug- injection of cGK activators into the presynaptic neuron can sub- gest possiblefunctions for this kinase in axonal outgrowth or stitute for postsynaptic neuron activity or NO synthesis (Arancio cellular differentiation of thesecells. Further support for this idea et al., 1995). Our studies demonstrating high densities of cGKI in derives from our observation of a transient expressionof cGKI in laminae I and II of the spinal cord suggest that similar retrograde the spinalfloor plate, a tissuethe inductive signalsof which specify 3136 J. Neurosci., May 15, 1996, 76(10):3130-3138 Qian et al. l cGMP-Dependent Protein Kinase in Dorsal Root Ganglioh

Figure 7. cGKI expression in embryonic spinal ganglia and floor plate.d, At E12, cGKI is specifically enriched in.DRG neurons (100X) and cells of the floor-plate (FP) and roof plate (B; 400X). Spinal neurons themselves are unstained, and weak labeling of blood vessels (BP’) is detected at this stage. C, D, Parasagittal sections of El5 embryos show that cGKI is enriched in a subset of DRG neurons and is present in both peripheral axons (Ax) and central projections to the dorsal spinal cord (DSC). E, F, At E15, nNOS is colocalized with cGKI in DRG and in axonal projections to the dorsal spinal cord (50X). Note that nNOS occurs in certain spinal neurons such as those of the intermediolateral column (IL) that lack cGKI. G, H, Both nNOS cGKI also colocalize in cutaneous branches of sensory axons such as those depicted here near the snout that derive from the trigeminal ganglia. Qian et al. l cGMP-Dependent Protein Kinase in Dorsal Root Ganglion J. Neurosci., May 15, 1996, 16(10):3130-3138 3137

Figure 9. G-substrate and cGKI are coexpressed in Purkinje cell soma. C, D, Immunoperoxidase staining of sagittal sections of rat cerebellum shows that G-substrate is specifically restricted to Purkinje (P) cell bodies and does not stain dendritic processes in the molecular layer (M) or axons in the granule cell layer (G). A, B, cGKI is also uniquely expressed in Purkinje cells but stains dendrites and axons as well as the cell body. E, F, nNOS shows a complimentary distribution to that of G-substrate and cGKl and is uniquely absent from Purkinje cells but, instead, stains granule cells in the granule cell layer and basket cells in the molecular layer.

REFERENCES Aanonsen LM, Wilcox GL (1987) Nociceptive action of excitatory amino acids in the mouse: effects of spinally administered opioids, phencycli- dine and sigma agonists. J Pharmacdl Exp Ther 243%19. Aimi Y. Fuiimura M. Vincent SR. Kimura H (1991) Localization of NADPH-diaphorase-containing neurons in sensory ganglia of the rat. J Comp Neurol306:382-392. Figure 8. G-substrate and cGKI arc colocalized in cerebellum and hip- Arancio 0, Kandel ER, Hawkins RD (1995) Activity-dependent long- pocampus. Immunohistochemistry demonstrates that (A) cGKI is ex- term enhancement of transmitter release by presynaptic 3’S’-cyclic pressed at highest levels in Purkinje cell processes of the molecular layer GMP in cultured hippocampal neurons. Nature 376:74-80. of the cerebellum (Cb). Lower levels of cGKI are apparent in dentate Aswad DW, Greengard P (1981) A specific substrate from rabbit cere- neurons and pyramidal cells of the hippocampus. B, G-substrate is also bellum for guanosine 3’:5’-monophosphate-dependent protein kinase. enriched in the cerebellum but is restricted to a linear layer of cells that I. Purification and characterization. J Biol Chem 256:3487-3493. correspond to Purkinje cell soma. The thalamus (r) contains G-substrate Bredt DS, Snyder SH (1990) Isolation of nitric oxide synthetase, a immunoreactivity but lacks cGKI. The cerebral cortex and the rest of the calmodulin-requiring enzyme. Proc Nat1 Acad Sci USA 87:682-685. forebrain are essentially devoid of cGKI or G-substrate. C, nNOS is also Bredt DS, Snyder SH (1994a) Nitric oxide: a physiologic messenger mol- most enriched in cerebellum but displays a broader forebrain distribution ecule. Annu Rev Biochem 63:175-195. than cGKl or G-substrate. Forty micrometer free-floating rat brain sec- Bredt DS, Snyder SH (1994b) Transient nitric oxide synthase neurons in tions were stained as described above. In these dark-field images, positive embryonic cerebral cortical plate, sensory ganglia, and olfactory epithe- labeling appears white. lium. Neuron 13:301-313. Bredt DS, Hwang PM, Snyder SH (1990) Localization of nitric oxide synthase indicating a neural role for nitric oxide. Nature 347:768-770. certain neuronal cell types (Yamada et al., 1991). Interestingly, Bredt DS, Glatt CE, Hwang PM, Fotuhi M, Dawson TM, Snyder SH protein kinase A has recently been noted to play a critical role in (1991) Nitric oxide synthase protein and mRNA are discretely localized in neuronal populations of the mammalian CNS together with NADPH specifying neuronal fate during Drosophila development (Li et al., diaphorase. Neuron 71615-624. 1995; Pan and Rubin, 1995). Disregulation of protein kinase A Chung K, Lee WT, Carlton SM (1988) The effects of dorsal rhizotomy also prevents floor plate-mediated differentiation of dopaminergic and spinal cord isolation on calcitonin gene-related peptide-labeled neurons of the substantia nigra in mammals (Hynes et al., 1995). terminals in the rat lumbar dorsal horn. Neurosci L&t 90:27-32. Coderre TJ, Yashpal K (1994) Intracellular messengers contributing to It will now be important to evaluate whether modulation of cGKI persistent nociception and hyperalgesia induced by L-glutamate and activity influences development and differentiation of sensory substance P in the rat formalin pain model. Eur J Neurosci neurons. 6:1328-1334. 3138 J Neuroscl, May 15, 1996, 16(10) 3130-3138 Qlan et al l cGMP-Dependent Protein Kmase m Dorsal Root Gangllon

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