- Email: [email protected]
Nitric oxide synthase and glutamate receptor immunoreactivity in the rat spinal trigeminal neurons expressing Fos protein after formalin injection
Brain Research 855 Ž2000. 107–115 www.elsevier.comrlocaterbres
Research report
Nitric oxide synthase and glutamate receptor immunoreactivity in the rat spinal trigeminal neurons expressing Fos protein after formalin injection a, )
Seng-Kee Leong a
, Hai-Ping Liu b , Jinn-Fei Yeo
b
Department of Anatomy, Faculty of Medicine, National UniÕersity of Singapore, Lower Kent Ridge Road, Singapore 119260, Singapore b Department of Oral and Maxillofacial Surgery, National UniÕersity of Singapore, Singapore 119260, Singapore Accepted 2 November 1999
Abstract Although recent studies implicated glutamate receptors and nitric oxide in nociception, much still needs to be known about their localisation in neurons involved in nociceptive transmission from the orofacial region. In this study, c-fos expression indicated by Fos immunohistochemistry in the caudal spinal trigeminal nucleus induced by subcutaneous injection of formalin into the lateral face of the rat was used as a marker for nociceptive neurons. The study sought to determine whether Fos-positive neurons express nitric oxide synthase, glutamate N-methyl-D-aspartate type receptor subunit 1, and glutamate alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid type receptor subunit 2r3; and whether they project to the thalamus. After formalin injection, many Fos-positive nuclei appeared in the superficial laminae of the ipsilateral trigeminal nucleus. Confocal laser scanning microscope revealed that almost all neurons with Fos immunofluorescent nuclei were colocalised with N-methyl-D-aspartate receptor 1, 94% with glutamate receptor 2r3 and 14% with nitric oxide synthase. Some of them were closely related to neurons labelled by nitric oxide synthase. Lastly, some of the Fos-positive neurons were labelled by tetramethylrhodamine-dextran injected into the trigeminothalamic tract or the thalamic region. The results suggested that activation of N-methyl-D-aspartate receptor 1 and glutamate receptor 2r3 upon glutamate release in response to noxious stimulation to the orofacial region might mediate c-fos expression in neurons involved in nociception. The expression of Fos in the neurons could also be mediated by nitric oxide produced from the same, as well as neighbouring neurons, when nociceptive stimulation persisted. Fos-positive neurons in the spinal trigeminal nucleus may project to the thalamus, relaying orofacial nociception to the higher sensory centre. q 2000 Elsevier Science B.V. All rights reserved. Keywords: Nociception; Colocalisation; Immunohistochemistry
1. Introduction Though the caudal spinal trigeminal nucleus ŽcSTN. has been implicated in nociceptive transmission, the neuroactive chemicals and connections of its neurons have not been studied in detail. This is due mainly to the fact that the neurons involved could not be precisely localised for the above studies. Recent investigation, however, demonstrated that noxious stimulation of the tooth pulp, or facial skin or after tooth extraction w9,50,53x or subcutaneous injection of formalin into the lateral face w23x caused c-fos expression in the cSTN. This led to the conclusion that the Fos-positive neurons are probably involved in nociception. The c-fos expressing neurons are present predominantly in lamina I, outer part of lamina II ŽIIo. and laminae V–VI of the spinal cord. These correspond to the terminal fields of
)
Corresponding author. Fax: q65-7787643; e-mail: [email protected]
primary nociceptive afferent fibres and to the distribution of nociresponsive neurons identified by electrophysiological recording w5,24,47x. In the case of the cSTN, they are also located predominantly in the superficial laminae of the nucleus. Hence, Fos, the nuclear protein encoded by the early gene c-fos, as demonstrated by immunohistochemistry, is a suitable indicator for the presence of nociceptive neurons activated by formalin injection into the lateral face. The formalin test as a means for assessing pain from the hindpaw, of upper lip or vibrissal pad of animals has, in fact, been widely used w7,14,36,56x. Recently, glutamate receptors and nitric oxide ŽNO. have been implicated in nociception and pain processing. In particular, activation of N-methyl-D-aspartate ŽNMDA. receptor has been shown to mediate sensitisation phenomena in spinal neurons following peripheral insult w55x. Spinal delivery of NMDA also produces hyperalgesia, an augmented response to noxious stimulation w1,8x. Moreover, this spinal hyperalgesia, as in the tonic phase of the
0006-8993r00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 6 - 8 9 9 3 Ž 9 9 . 0 2 3 1 6 - 1
108
S.-K. Leong et al.r Brain Research 855 (2000) 107–115
formalin test, is blocked by intrathecal injection of NMDA antagonists w38x. Inhibition of spinal NO synthesis blocks NMDA-induced hyperalgesia w29,39x, and thermal hyperalgesia resulting from nerve injury w41x. It also produces antinociception in the rat injected with formalin at the paw w49x. In addition to NMDA receptor, other types of glutamate receptors, the metabotropic and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid ŽAMPA. receptors have also been implicated in nociception w35,57x. Although glutamate receptors and NO have been widely reported to play important roles in spinal pain processing, very little is known in the case of orofacial nociception which is transmitted via afferent fibres of trigeminal ganglion neurons to the cSTN. From here, information is conveyed to the thalamus w52x then to the cerebral cortex. The present study aimed first to investigate the distribution of neurons in the cSTN that expressed Fos protein in response to subcutaneous formalin injection into the lateral face of the rat. This was followed by studying whether Ži. these neurons express glutamate receptors or NO synthase ŽNOS.; Žii. they project to the thalamus. The retrograde axonal tracer, tetramethylrhodamine-dextran ŽTMR-D., was injected into the thalamus or trigeminothalamic tract to reveal the trigeminothalamic neurons. Our preliminary study indicated that of all the glutamate receptors, NMDA receptor subunit 1 ŽNMDAR1. and AMPA receptor subunit 2r3 ŽGluR2r3. showed the most intense immunostaining in the neurons of the cSTN and should therefore play the most prominent role in nociception. Subsequently, the investigation focused on the two receptors.
2. Materials and methods A total of 24 adult male Wistar rats weighing between 250 and 300 g were used in the present study. Animal treatments and maintenance confirmed to the guidelines of the International Association for the Study of Pain w58x. The animals were divided into three groups. Group 1 consisted of 10 rats that received subcutaneous injection of dilute formalin into the lateral face only. Group 2 consisted of 10 rats which received a combination of thalamic or trigeminothalamic tract injection of TMR-D and formalin stimulation. After TMR-D injection, the rats were allowed to survive for 5–7 days and were perfused transcardially at 2 h after formalin injection. There were four rats in Group 3; these received saline injection only. Since our preliminary study showed that NMDAR1 and GluR2r3 staining in the superficial laminae of the cSTN were the strongest among all the NMDA and AMPA receptors, NMDAR1 and GluR2r3 were used for the colocalisation study. 2.1. Thalamic or trigeminothalamic tract injection TMR-D Ž3000 MW, anionic, lysing fixable; Molecular Probe, Eugene, OR. was dissolved in 0.1 M citrate-NaOH
at pH 3.0 and made up to 10% solution w26x. Anaesthesia of the rats was achieved by i.p. injection of chloral hydrate Ž35 mgrkg b.wt... Usually, no more than 7 mgrkg b.wt. was needed to maintain anaesthesia. The anaesthetised rats were placed on a stereotaxic apparatus and TMR-D solution was injected through a 5 ml microsyringe ŽExmire, 33 gauge. inserted into the right thalamic region or right trigeminothalamic tract. Injection was made according to the coordinates in the atlas by Paxinos and Watson w46x. In six rats, a total of 0.4–0.8 ml TMR-D solution was injected into the thalamic region. In each rat, the solution was injected into four regions: paracentral thalamic nucleus, posterior thalamic nuclear group, ventral posterolateral thalamic nucleus and ventral posteromedial thalamic nucleus. In four rats, 0.5 ml TMR-D solution was delivered to the right trigeminothalamic tract. Each dose of TMR-D was delivered within an interval of 10 min, with the needle of the syringe left in place for another 10 min. 2.2. Lateral face injection Under chloral hydrate anaesthesia, 0.5 ml of 4% formalin in saline was injected subcutaneously into multiple sites of the left lateral face of anaesthetised rats with a 0.5-ml syringe. Formalin injections were delivered to all Group 1 normal rats and Group 2 rats that had earlier received TMR-D injection into the right thalamic region or trigeminothalamic tract. Group 3 control rats received subcutaneous injection of 0.5 ml of physiological saline in the same manner as those receiving formalin injection. 2.3. Perfusion and tissue sectioning All the rats were anaesthetised with an overdose of chloral hydrate and perfused with 100 ml of Ringer’s solution 2 h after formalin or saline injection into the lateral face, followed by 500 ml of 0.1 M phosphate-buffer ŽpH 7.4. containing 4% Žwrv. paraformaldehyde. The caudal medulla and cervical spinal cord was then removed from the obex to the C2 segment and post-fixed in the same fixative for 2 h. Then the tissue was soaked in 20% sucrose in 0.1 M phosphate buffer overnight. Frozen sections were cut at 30 mm thickness in a cryostat ŽJung Frigocut 2800E. at y158C, and collected in a series of one in four sections in wells filled with phosphate buffered saline and 0.3% Triton X-100 ŽPBS-TX, pH 7.4.. 2.4. Immunohistochemistry and Õisualization of labelling 2.4.1. Fos immuno-DAB reaction or immunofluorescence Frozen sections for the detection of Fos protein product only were processed for Fos immuno-DAB reaction. They were washed for 2 h in 0.1 M PBS containing 0.3% Triton X-100, then blocked with 5% normal rabbit serum in PBS for half an hour. Polyclonal sheep anti-Fos ŽChemicon
S.-K. Leong et al.r Brain Research 855 (2000) 107–115
International, USA. were then applied on the sections overnight at a dilution of 1:1000 in 0.1 M PBS, containing 0.1% Triton X-100. After incubation, the sections were washed in 0.1 M PBS and placed in biotinylated rabbit anti-sheep IgG Ž1:200; Vector. for 1 h and washed again in 0.1 M PBS, followed by treatment with avidin–biotin–peroxidase complex ŽVector. for 1 h, and finally washed three times in 0.1 M PBS. Fos-LI was visualized with incubation in a nickel diaminobenzidine solution activated by 0.005% hydrogen peroxide for 3–4 min. Sections were then mounted on clean gelatin-coated slides and air-dried. They were dehydrated and mounted with permount and covered with coverslips. For Fos immunofluorescence, the sections were washed in 0.1 M PBS, after incubation with polyclonal sheep anti-Fos overnight, placed in donkey antisheep fluorescein conjugated IgG ŽChemicon International, USA. at a dilution of 1:160 for 1 h, and finally washed three times in 0.1 M PBS. After Fos immunofluorescence,
109
sections from rats receiving TMR-D injection into the thalamus or trigeminothalamic tract were collected on gelatin-coated slides, mounted with DAKO fluorescent mounting medium and covered with coverslips. Sections for double immunolabelling were stained for NOS, GluR2r3 and NMDAR1 immunofluorescence later. 2.4.2. Double labelling of Fos with NOS, GluR2r 3 and NMDAR1 After staining for Fos immunofluorescence, the sections that would be further processed for NOS, NMDAR1 or GluR2r3 immunofluorescence were incubated with polyclonal rabbit anti-NOS ŽChemicon International, USA, 1:500., or polyclonal rabbit anti-NMDAR1 ŽChemicon International, USA, 1 mr1 ml., or polyclonal rabbit antiGluR2r3 ŽChemicon International, USA, 1 mr1 ml.. After incubation, they were washed in 0.1 M PBS, and placed in cyanine 3 conjugated donkey anti-rabbit IgG ŽChemicon
Fig. 1. Photomicrograph of Fos labelling in the caudal medulla of the rat receiving formalin injection into the left lateral face. Note that nuclei intensively labelled by Fos immunohistochemistry are mostly concentrated in lamina I and the outer part of lamina II ŽIIo. of the left caudal spinal trigeminal nucleus ŽcSTN. with a few of them also scattered in the deeper laminae. Scale bar s 200 mm. Fig. 2. Photomicrograph of Fos labelling in the caudal medulla of the rat receiving physiological saline injection into the left lateral face. Note that the number of positive nuclei and their staining intensity is much less than that in Fig. 1. Scale bar s 200 mm.
110
S.-K. Leong et al.r Brain Research 855 (2000) 107–115
S.-K. Leong et al.r Brain Research 855 (2000) 107–115
International, USA. at a dilution of 1:600 for 1 h, and finally washed three times in PBS. They were then collected on gelatin-coated slides, mounted with DAKO fluorescent mounting medium and covered with coverslips. Control sections were incubated without primary antibodies. 2.4.3. Double labelling of TMR-D with Fos or NOS Sections from rats receiving TMR-D injection into the thalamus or trigeminothalamic tract were processed for Fos or NOS immunofluorescence as mentioned previously. They were then collected on gelatin-coated slides, mounted with DAKO fluorescent mounting medium and covered with coverslips. 2.4.4. Visualization of double labelling with confocal scanning microscopy Sections were examined with a Carl Zeiss, LSM410 confocal laser scanning microscope. Each section was initially scanned with a 488 nm laser line, and an emission filter BP 510–525, for the detection of fluorescein; and with a 543-nm laser line, and an emission filter LP 570, for the detection of cyanine 3 or TMR-D. 2.4.5. Cell counting To study the distribution of Fos-positive neurons in the cSTN, Fos-labelled nuclei were counted in 10 sections per rat through the main part of the left cSTN. To obtain the percentage of Fos-positive neurons colocalised with NMDAR-1 and GluR2r3, NOS or TMR-D in the cSTN, the cells were counted from 12 sections per rat. Sections for double staining of Fos with NMDAR-1 or GluR2r3 were collected from the same four rats while those for Fos and NOS double labelling were collected from another four rats as the rabbit anti-NOS was not available until a later date.
111
3. Results 3.1. Distribution of Fos-positiÕe neurons in the cSTN Fos labelling was observed in neuronal nuclei. In 18 rats receiving formalin injection into the left lateral face, an abundance of labelled nuclei was distributed mainly in lamina I, and the outer part of lamina II ŽIIo.. Some of them were also seen in the deeper laminae of the cSTN ŽFig. 1. ipsilateral to the side of formalin injection Žleft.. Counted from four rats, the number of Fos-positive neurons throughout the laminae of the left cSTN in one section ranged from 50 to 135. Fos-positive neurons located in laminae I and IIo ranged from 36 to 109 per section. There were very few labelled Fos-positive neurons in the deeper laminae of the nucleus, and such were less intensely labelled. In rats receiving physiological saline injection, much fewer Fos-positive neurons were seen in laminae I and IIo of the cSTN ŽFig. 2. ipsilateral to the side of injection, compared to the formalin injected ones. These were much less intensely stained. 3.2. Colocalisation of Fos with other neuroreactiÕe chemicals in the cSTN In eight rats receiving only formalin injection into the left lateral face, double immunofluorescence was used to detect whether Fos-positive neurons would also express glutamate receptor subunits and NOS. The sections incubated without primary antibodies were not stained. Counted from four rats, almost all the Fos-positive neurons were NMDAR1-positive ŽFig. 3.. 1272 of 1274 Fos-positive neurons were NMDAR1-positive. Most Fos-positive neurons were GluR2r3-positive ŽFig. 4.. A total of 889 out of 943, i.e., about 94% of Fos-positive neurons, were GluR2r3-positive. Only 14% of Fos-positive neurons Ži.e.,
Fig. 3. Photomicrograph shows that Fos-positive neurons induced by formalin injection are colocalised with NMDAR1 immunofluorescence Žarrows. in the superficial laminae of the cSTN. Note that the Fos-positive nuclei Žgreen. are surrounded by NMDAR1-positive cytoplasm Žred.. Fig. 4. Photomicrograph shows that Fos-positive neurons induced by formalin injection are colocalised with GluR2r3 immunofluorescence Žarrowed. in the superficial laminae of the cSTN. Note that the Fos-positive nuclei Žgreen. are surrounded by GluR2r3-positive cytoplasm Žred.. Fig. 5. Photomicrograph showing double labelling of Fos and NOS in the left cSTN of a rat receiving formalin injection into the left lateral face. Note that neurons containing nuclei labelled by Fos Žgreen. are more superficially located than those labelled by NOS Žred.. Note also that two Fos-positive nuclei are surrounded by NOS-positive cytoplasm Žarrow and arrowhead.. The insert shows a magnified view of the neuron indicated by an arrowhead. Fig. 6. Three Fos-labelled nuclei Žgreen. in the superficial laminae of the cSTN are shown in this photomicrograph. Two of them Žarrowed. are closely related to NOS-positive neurons Žred.. Fig. 7. Photomicrograph showing an NOS-positive neuron Žarrowed. with two processes Žred. in the superficial laminae of cSTN, one of which extends to the close vicinity of a neuron bearing a Fos-positive nucleus Žgreen.. Fig. 8. A TMR-D filled neuron Žarrowed. Žcytoplasm in red. contains a Fos-positive nucleus Žgreen. together with a few Fos-positive nuclei in the superficial laminae of the left cSTN in a rat receiving formalin injection in the left lateral face 5 days after TMR-D injection into the right trigeminothalamic tract. Insert shows the higher magnification of the neuron indicated by an arrow.
112
S.-K. Leong et al.r Brain Research 855 (2000) 107–115
48 of 354. were NOS-positive ŽFig. 5.. However, some Fos-labelled nuclei were observed to lie close to NOSpositive neuronal cell bodies ŽFig. 6. or neurites ŽFig. 7. in lamina IIo. 3.3. TMR-D labelled trigeminothalamic neurons The injection of the tracer TMR-D was targeted at the right trigeminothalamic tract or thalamus and the injection sites were confirmed by examination of the sections of the brain through the injected plane. In six rats receiving right thalamic injection and four rats receiving right trigeminothalamic tract injection of TMR-D, many neurons in the left cSTN were labelled by TMR-D. There were more labelled neurons in the deeper laminae than in laminae I and II. More labelled neurons were detected in the cSTN after trigeminothalamic tract injection than after thalamic injection of TMR-D. After formalin stimulation, TMR-D injected rats showed colocalisation of TMR-D with some Fos-positive cells in laminae I and II of the cSTN ŽFig. 8.. Counted from two rats receiving trigeminothalamic tract injection, 169 neurons were labelled with TMR-D. Only six TMR-D labelled neurons were Fos-positive in laminae I and II of the cSTN in the two rats. None of them were NOS-positive.
4. Discussion The present study showed that many Fos-positive nuclei appeared in the superficial laminae of the cSTN within 2 h of subcutaneous formalin injection into the lateral face of the rat. Most Fos-positive neurons expressed glutamate NMDA receptor subtype 1 ŽNMDAR1. and AMPA receptor subtype, GluR2r3. Only 14% of Fos-positive neurons expressed NOS, but some Fos-positive cells were found in close apposition to NOS-positive neurites and cell bodies. Lastly, some of the Fos-positive neurons were labelled by the neuronal tracer, TMR-D, injected into the trigeminothalamic tract or thalamic region. 4.1. Fos expression in the cSTN The cSTN is an important component in the relay of sensory information to higher brain centres w28x. A large number of studies have demonstrated that primary afferent fibres from the orofacial region terminate within the STN w17,44,45x and that laminae I and II of the STN, which receive small-diameter trigeminal afferent fibres, are primarily responsible for relaying orofacial non-reflexive nociceptive information w4x. The detection of the proto-oncogene c-fos and its protein product Fos has been regarded as a valuable tool for pain research w24x. Though not all Fos expressing neurons could be regarded as nociceptive neurons w20x, there is enough evidence to show that subcutaneous injection of
formalin can evoke profound hyperalgesia w56x and that intrathecally administered c-fos antisense oligodeoxynucleotides to the rat prior to formalin injections dose dependently decreased both Fos immunoreactivity in the dorsal horn of the rat lumbar cord and all nociceptive measures in the tonic phase of the formalin test w23x. Thus, Fos can be considered a reliable indirect marker for nociceptive neurons. The test for pain induced by formalin injection into the rat paw has gained wide recognition. Subcutaneous injection of formalin into the orofacial region has also been generally accepted as a reliable method in the study of orofacial pain w7,14x. Our finding that Fos-positive nuclei were principally localised in laminae I and IIo of the cSTN after subcutaneous injection of formalin into the lateral face of the rat is consistent with other observations on the cSTN after formalin administration into the lateral face, lips or supra-ocular region w25,33,54,30,51x. This also corresponds to the distribution of Fos-positive neurons in the superficial laminae of the spinal cord after cutaneous formalin stimulation in the hindpaw w24,47,5x. In this study, we observed that the number of Fos-positive neurons induced in the cSTN by formalin injection into the lateral face was much more than that induced by saline injection. Taking all this together, it is reasonable to surmise that though not all the Fos expressing neurons in the superficial laminae of the cSTN could be considered nociceptive neurons, as Fos expression may be induced by other forms of stimuli w20x, the large majority of them should be so regarded. On the other hand, it is critical to recognise that the absence of c-fos expression cannot be taken to mean an absence of neural activity w12x. 4.2. Colocalisation of Fos and NMDAR1 and GluR2r 3 in the neurons of the cSTN Many endogenous substances have been implicated in the genesis of pain. In addition to neuropeptides like substance P and calcitonin gene-related peptide, which are released in the spinal cord in vivo upon noxious peripheral stimulation w16,13,42x, glutamate through its receptor of the NMDA type has also been implicated in the production of the hyperalgesic state w56x. Activation of NMDA receptors causes release of substance P in the spinal cord w37x. Antagonists of NMDA receptors inhibit wind-up and other manifestations of spinal hyperexcitability w19,48,55x. Additionally, glutamate receptor of the NMDA type contributes significantly to the long-term change in central nociceptive neurons that accompany prolonged noxious chemical stimulation w19,43x. In the case of non-NMDA Žincluding AMPA and kainate. receptors, they most likely mediate the short-term activation of second-order neurons by peripheral nociceptors as well as other sensory receptors. Antagonism of non-NMDA receptors blocks the response of second-order neurons to all peripheral stimuli w11x. Non-NMDA receptors also contribute to all stages of
S.-K. Leong et al.r Brain Research 855 (2000) 107–115
hyperalgesia induced by inflammation w43x. The present results suggest an involvement of both NMDA and AMPA receptors in the short-term processing of inputs from nociceptive afferents at the level of the cSTN 2 h after formalin injection. The fact that NMDAR1 and GluR2r3 were expressed in most Fos-positive neurons in the superficial laminae of the cSTN, suggested that activation of both receptors upon glutamate release in response to noxious stimulation might mediate c-fos expression in neurons involved in hyperalgesia. This is supported by several lines of evidence obtained from pharmacological experiments, using subcutaneous injection of chemical irritants such as formalin or carrageenin as the noxious stimulation. These showed that intrathecal or systemic administration of NMDA receptor antagonists reduced the number of Fos-positive spinal neurons w6,27x or the expression of c-fos mRNA w15x, compared to controls. There was also correlation in the time course of the appearance of nociceptive behavioural signs and c-fos mRNA labelling w15x. No similar time course correlation has been performed in the study of orofacial pain induced by formalin injection into the face region. 4.3. Fos expressing neurons and NOS-positiÕe neurons in the cSTN In addition to glutamate receptors, recent studies reported that NO, a novel neurotransmitter in the nervous system w3x synthesized by NOS, could also play a role in nociceptive processing and could be involved in the production of a hyperalgesic state w18,39,40x. Pretreatment with intrathecal doses of NO inhibitor, N v-nitro-L-arginine methyl ester ŽL-NAME., significantly reduced the licking behavior associated with the injection of formalin into the left hindpaw of the rat w49x. It also reduced Fos labelling in the spinal cord induced by noxious mechanical or chemical irritant stimulation w32,22,49x. These two observations pointed clearly to a role of NO in c-fos expression caused by noxious stimulation. Thus, the colocalisation of Fos and NOS in the same neurons in the superficial laminae of the cSTN in response to subcutaneous formalin injection into the lateral face should be expected. The results indicated that only 14% of Fos-positive cSTN neurons expressed NOS immunoreactivity while the majority of NOS-positive neurons were Fos-negative. While Fos expression indicates activation of neurons in response to nociceptive stimulation, the absence of it in the NOS-positive neurons may not indicate that these neurons are not activated as a previous study has shown that not all neurons express the gene when activated w12x. The observation that some Fos-positive cSTN neurons were seen in close apposition to NOS-positive neuronal cell bodies as well as neuronal profiles is mirrored by that of Lee et al. w31x in the dorsal horn of the lumbar cord. Since NO is a gaseous molecule and is able to diffuse
113
through cell membranes freely, this neighbouring relationship suggests that NO might modulate the function of Fos-positive neurons via a paracrine mode. NO can diffuse from neuronal cell bodies or dendritic or axonal processes of these cells to adjacent neurons. Hence, NO may act on Fos-positive neurons through both intracellular as well as extracellular pathways to activate guanylyl cyclase, which in turn activates the cGMP pathway that may ultimately induce Fos expression and further bring about nociception. In addition to modulating Fos expression induced by nociception, NO may also modulate other functions as colocalisation of Fos with NOS was found in many oxytocin and vasopressin synthesizing neurons of the rat hypothalamus following stress stimuli w21x. 4.4. Fos expressing neurons projecting to the thalamus Of the TMR-D labelled neurons in the superficial laminae of the cSTN following injection of TMR-D injection into the right trigeminothalamic tract or thalamus, some were Fos-positive. These were obviously thalamic projecting neurons. This observation is consistent with that of Li et al. w33,34x who showed Fos expression in the cSTN after subcutaneous injection of formalin into the lips of rats. Since almost all Fos-positive neurons in the cSTN were NMDAR1-positive as demonstrated in this study, it is clear that some Fos and NMDAR1 coexpressing neurons in the cSTN were neurons projecting to the thalamus. They may relay orofacial nociception to the higher sensory centre. That not many TMR-D labelled neurons were found in the cSTN in the present study could be partially accounted for by the fact that some of the nociceptive neurons could project to other nuclei in the brain. For example, it had been demonstrated that formalin injection into the perioral region of the rat induced Fos expression in the parabrachial nucleus w59x and that formalin induced Fos-positive neurons in the cSTN projected to the parabrachial nucleus w54,33x. Thus, in addition to the thalamus, other nuclei may play important roles in conveying nociception from the orofacial region to the higher sensory centre. Significantly, none of the NOS-positive neurons were colocalised with TMR-D labelled ones, indicating that these neurons did not project to the thalamus. This is consistent with the finding of Dohrn et al. w10x. It may, therefore, be concluded that NOS-positive neurons in the superficial laminae of the cSTN are not projecting neurons. They may play a role in the local circuit for nociception as suggested by Aimar et al. w2x that NO producing neurons modulate the release of sensory neuropeptides in the rat substantia gelatinosa of the dorsal horn.
Acknowledgements We wish to thank Professor Lawrence Kruger from the Department of Neurobiology, School of Medicine, UCLA
114
S.-K. Leong et al.r Brain Research 855 (2000) 107–115
for his valuable comments on the manuscript. This work was supported by grants RP940320 and RP960331 from the National University of Singapore. References w1x L.M. Aanonsen, G.L. Wilcox, Nociceptive action of excitatory amino acids in the mouse: effects of spinally administered opioids, phencyclidine and sigma agonists, J. Pharmacol. Exp. Ther. 243 Ž1987. 9–19. w2x P. Aimar, L. Pasti, G. Carmignoto, A. Merighil, Nitric oxide-producing islet cells modulate the release of sensory neuropeptides in the rat substantia gelatinosa, J. Neurosci. 18 Ž1998. 10375–10388. w3x D.S. Bredt, S.H. Snyder, Nitric oxide, a novel neuronal messenger, Neuron 8 Ž1992. 3–11. w4x J.G. Broton, J.W. Hu, B.J. Sessle, Effects of temporomandibular joint stimulation on nociceptive and nonnociceptive neurons of the cat’s trigeminal nucleus caudalis Žmedullary dorsal horn., J. Neurophysiol. 59 Ž1988. 575–1589. w5x E. Bullitt, Somatotopy of spinal nociceptive processing, J. Comp. Neurol. 312 Ž1991. 279–290. w6x V. Chapmann, P. Honore, J. Buritova, J.M. Besson, The contribution of NMDA receptor activation to spinal c-fos expression in a model of inflammatory pain, Br. J. Pharmacol. 116 Ž1995. 1628–1634. w7x P. Clavelou, J. Pajot, R. Dallel, P. Raboisson, Application of the formalin test to the study of orofacial pain in the rat, Neurosci. Lett. 103 Ž1989. 349–353. w8x T.J. Coderre, R. Melzack, The distribution of excitatory amino acids to central sensitization and persistent nociception after formalin-induced tissue injury, J. Neurosci. 12 Ž1992. 3665–3670. w9x F. Coimbra, A. Coimbra, Dental noxious input reaches the subnucleus caudalis of the trigeminal complex in the rat, as shown by c-fos expression upon thermal or mechanical stimulation, Neurosci. Lett. 173 Ž1994. 201–204. w10x C.S. Dohrn, M.A. Mullett, R.H. Price, A.J. Beitz, Distribution of nitric oxide synthase-immunoreactive interneurons in the spinal trigeminal nucleus, J. Comp. Neurol. 346 Ž1994. 449–460. w11x P.M. Dougherty, J. Palecek, V. Paleckova, L.S. Sorkin, W.D. Willis, The role of NMDA and non-NMDa excitatory amino acid receptors in the excitation of primate spinothalamic tract neurons by mechanical, chemical, thermal, and electrical stimulation, J. Neurosci. 12 Ž1992. 3014–3025. w12x M. Dragunow, R. Faull, The use of c-fos as a metabolic marker in neuronal pathway tracing, J. Neurosci. Methods 29 Ž1989. 261–265. w13x A.W. Duggan, I.A. Hendry, C.R. Morton, W.D. Hutchison, Z.Q. Zhao, Cutaneous stimuli releasing immunoreactive substance P in the dorsal horn of the cat, Brain Res. 451 Ž1988. 261–271. w14x E. Eisenberg, B.P. Vos, A.M. Strassman, The NMDA antagonist memantine blocks pain behavior in a rat model of formalin-induced facial pain, Pain 54 Ž1993. 301–307. w15x K.J. Elliott, M. Brodsky, A.D. Hynansky, K.M. Foley, C.E. Inturrisi, Dextromethorphan suppresses both formalin-induced nociceptive behavior and the formalin induced increase in spinal cord c-fos mRNA, Pain 61 Ž1995. 401–409. w16x V.L. Go, T.L. Taksh, Release of substance P from the cat spinal cord, J. Physiol. 391 Ž1987. 141–167. w17x F. Greenwood, An electrophysiological study of the central connections of primary afferent nerve fibres from the dental pulp in the cat, Arch. Oral Biol. 18 Ž1973. 771–785. w18x J.E. Haley, A.H. Dickenson, M. Schachter, Electrophysiological evidence for a role of nitric oxide in prolonged chemical nociception in the rat, Neuropharmacology 31 Ž1992. 251–258. w19x J.E. Haley, A. Sullivan, A. Dickenson, Evidence for spinal Nmethyl-D-aspartate receptor involvement in prolonged chemical nociception in the rat, Brain Res. 518 Ž1990. 218–226.
w20x J.A. Harris, Using c-fos as a neural marker of pain, Brain Res. Bull. 45 Ž1998. 1–8. w21x S. Hatakeyama, Y. Kawai, T. Ueyama, E. Senba, Nitric oxide synthase-containing magnocellular neurons of the rat hypothalamus synthesize oxytocin and vasopressin and express Fos following stress stimuli, J. Chem. Neuroanat. 11 Ž1996. 243–256. w22x P. Honore, V. Chapman, J. Buritova, J.M. Besson, Reduction of carrageenin oedema and the associated c-fos expression in the rat lumbar spinal cord by nitric oxide synthase inhibitor, Br. J. Pharmacol. 114 Ž1995. 77–84. w23x W.Y. Hou, B.C. Shyu, T.M. Chen, J.W. Lee, J.Y. Shieh, W.Z. Sun, Intrathecally administered c-fos antisense oligodeoxynucleotide decreases formalin-induced nociceptive behavior in adult rats, Eur. J. Pharmacol. 329 Ž1997. 17–26. w24x S.P. Hunt, A. Pini, G. Evan, Induction of c-fos-like protein in spinal cord neurons following sensory stimulation, Nature 328 Ž1987. 632–634. w25x K. Iwata, K. Kanda, Y. Tsuboi, K. Kitajima, R. Sumino, Fos induction in the medullary dorsal horn and C1 segment of the spinal cord by acute inflammation in aged rats, Brain Res. 678 Ž1995. 127–139. w26x T. Kaneko, K. Saeki, T. Lee, N. Mizuno, Improved retrograde axonal transport and subsequent visualization of tetramethylrhodamine ŽTMR.-dextran amine by means of an acidic injection vehicle and antibodies against TMR, J. Neurosci. Methods 65 Ž1996. 157–165. w27x L.J. Kehl, K.R. Gogas, L. Lichtblau, C.H. Pollock, M. Mayes, A.I. Basbaum, G.L. Wilcox, The NMDA antagonist MK801 reduces noxious stimulus induced FOS expression in the spinal cord dorsal horn, in: M.R. Bond, J.E. Charlton, C.J. Woolf ŽEds.., Proceedings of the 6th World Congress on Pain, Elsevier, New York, 1991, pp. 307–331. w28x J.P. Kelly, Trigeminal system, in: E.R. Kandel, J.H. Schwartz ŽEds.., Principles of Neural Science, Elsevier, New York, 1986, pp. 381–393. w29x K.F. Kitto, J.E. Haley, G.W. Wilcox, Involvement of nitric oxide in spinally mediated hyperalgesia in the mouse, Neurosci. Lett. 148 Ž1992. 1–5. w30x A. Laudanna, M.I. Nogueira, M. Mariano, Expression of Fos protein in the rat central nervous system in response to noxious stimulation: effects of chronic inflammation of the superior cervical ganglion, Braz. J. Med. Biol. Res. 31 Ž1998. 847–850. w31x J.H. Lee, R.H. Price, F.G. Williams, B. Mayer, A.J. Beitz, Nitric oxide synthase is found in some spinothalamic neurons and neurons processes that appose spinal neurons that express Fos induced by noxious stimulation, Brain Res. 608 Ž1993. 324–333. w32x J.H. Lee, G.L. Wilcox, A.J. Beitz, Nitric oxide mediates Fos expression in the spinal cord induced by mechanical noxious stimulation, Neurol. Report 3 Ž1992. 841–844. w33x J.L. Li, T. Kaneko, S. Nomura, Y.Q. Li, N. Mizuno, Association of serotonin-like immunoreactive axon with nociceptive projection neurons in the caudal spinal trigeminal nucleus of the rat, J. Comp. Neurol. 384 Ž1997. 127–141. w34x J.L. Li, Y.Q. Li, T. Kaneko, N. Mizuno, Preprodynorphin-like immunoreactivity in medullary dorsal horn neurons projecting to the thalamic regions in the rat, Neurosci. Lett. 264 Ž1999. 13–16. w35x S. Maione, I. Marabese, J. Leyva, E. Palazzo, V. de Novellis, F. Rossi, Characterisation of mGluRs which modulate nociception in the PAG of the mouse, Neuropharmacology 37 Ž1998. 1475–1483. w36x Y. Mineta, E. Eisenberg, A.M. Strassman, Distribution of Fos-like immunoreactivity in the caudal medullary reticular formation following noxious facial stimulation in the rat, Exp. Brain Res. 107 Ž1995. 34–38. w37x H.T. Liu, P.W. Mantyh, A.I. Basbaum, NMDA-receptor regulation of substance P release from primary afferent nociceptors, Nature 386 Ž1997. 721–724. w38x A.B. Malmberg, T.L. Yaksh, Hyperalgesia mediated by spinal gluta-
S.-K. Leong et al.r Brain Research 855 (2000) 107–115
w39x
w40x w41x
w42x
w43x
w44x w45x
w46x w47x
w48x w49x
mate or SP receptor blocked by spinal cyclooxygenase inhibition, Science 247 Ž1992. 1276–1279. A.B. Malmberg, T.L. Yaksh, Spinal nitric oxide synthesis inhibition blocks NMDA-induced thermal hyperalgesia and produced antinociception in the formalin test in rats, Pain 54 Ž1993. 291–300. S.T. Meller, G.F. Gebhart, Nitric oxide ŽNO. and nociceptive processing in the spinal cord, Pain 52 Ž1993. 127–136. S.T. Meller, P.S. Pechman, G.F. Gebhart, T.J. Maves, Nitric oxide mediates the thermal hyperalgesia produced in a model of neuropathic pain in the rat, Neuroscience 50 Ž1992. 7–10. C.R. Morton, W.D. Hutchison, Release of sensory neuropeptides in the spinal cord: study with calcitonin gene-related peptide and galanin, Neuroscience 31 Ž1989. 807–815. V. Neugebauer, T. Lucke, H.G. Schaible, N-methyl-D-aspartate ŽNMDA. and non-NMDA receptor antagonists block the hyperexcitability of dorsal horn neurons during development of acute arthritis in rat’s knee joint, J. Neurophysiol. 70 Ž1993. 1365–1377. S.G. Nord, Responses of neurons in rostral and caudal trigeminal nuclei to tooth pulp stimulation, Brain Res. Bull. 1 Ž1976. 489–492. W.M. Panneton, H. Burton, Corneal and periocular representation within the trigeminal sensory complex in the cat studied with transganglionic transport of horseradish peroxidase, J. Comp. Neurol. 199 Ž1981. 327–344. G. Paxinos, C. Watson, The Rat Brain in Stereotaxic Coordinates, 3rd edn., Academic Press, San Diego, 1996. R.W. Presley, D. Menetrey, J.D. Levine, A.I. Basbaum, Systemic ´ morphine suppresses noxious stimulus-evolked fos protein-like immunoreactivity in the rat spinal cord, J. Neurosci. 10 Ž1990. 323–335. K. Ren, Wind-up and the NMDA receptor: from animal studies to humans, Pain 59 Ž1994. 157–158. A.K. Rocher, M. Cook, G.L. Wilcox, K.C.A. Kajander, A nitric oxide synthesis inhibitor ŽL-NAME. reduces licking behavior and Fos-labelling in the spinal cord of rats during formalin-induced inflammation, Pain 66 Ž1996. 331–341.
115
w50x A.M. Strassman, B.P. Vos, Y. Mineta, S. Naderi, D. Borsook, R. Burstein, Fos-like immunoreactivity in the superficial medullary dorsal horn induced by noxious and innocuous thermal stimulation of facial skin in the rat, J. Neurophysiol. 70 Ž1993. 1811–1821. w51x R. Terayama, N. Nagamatsu, T. Ikeda, T. Nakamura, O.I. Rahman, S. Sakoda, R. Shiba, T. Nishimori, Differential expression of Fos protein after transection of the rat infraorbital nerve in the trigeminal nucleus caudalis, Brain Res. 768 Ž1997. 135–146. w52x P.M.E. Waite, D.J. Tracey, Trigeminal sensory system, in: G. Paxinos ŽEd.., The Rat Nervous System, Academic Press, New York, 1995, pp. 705–724. w53x S. Wakisaka, Y. Sasaki, H. Ichikawa, S. Matsuo, Increase in c-foslike immunoreactivity in the trigeminal nucleus complex after dental treatment, Proc. Finn. Dent. Soc. 88 Ž1992. 551–555. w54x L.G. Wang, H.M. Li, J.S. Li, Formalin induced FOS-like immunoreactive neurons in the trigeminal spinal caudal subnucleus project to contralateral parabrachial nucleus in the rat, Brain Res. 649 Ž1994. 62–70. w55x C.J. Woolf, S.W.N. Thompson, The induction and maintenance of central sensitization is dependent on N-methyl-D-aspartic acid receptor activation: implications for the treatment of post-injury pain hypersensitivity states, Pain 44 Ž1991. 293–300. w56x T.L. Yaksh, A.B. Malmberg, Central pharmacology of nociceptive transmission, in: P.D. Wall, R. Melzack ŽEds.., The Textbook of Pain, Churchill-Livingstone, Edinburgh, 1994, pp. 165–200. w57x P.K. Zahn, E. Umali, T.J. Brennan, Intrathecal non-NMDA excitatory amino acid receptor antagonists inhibit pain behaviors in a rat model of postoperative pain, Pain 74 Ž1998. 213–223. w58x M. Zimmermann, Ethical guidelines for investigation of experimental pain in conscious animals, Pain 16 Ž1983. 109–110. w59x O. Hermanson, A. Blomqvist, Subnuclear localization of FOS-like immunoreactivity in the parabrachial nucleus after orofacial nociceptive stimulation of the awake rat, J. Comp. Neurol. 387 Ž1997. 114–123.
Research report
Nitric oxide synthase and glutamate receptor immunoreactivity in the rat spinal trigeminal neurons expressing Fos protein after formalin injection a, )
Seng-Kee Leong a
, Hai-Ping Liu b , Jinn-Fei Yeo
b
Department of Anatomy, Faculty of Medicine, National UniÕersity of Singapore, Lower Kent Ridge Road, Singapore 119260, Singapore b Department of Oral and Maxillofacial Surgery, National UniÕersity of Singapore, Singapore 119260, Singapore Accepted 2 November 1999
Abstract Although recent studies implicated glutamate receptors and nitric oxide in nociception, much still needs to be known about their localisation in neurons involved in nociceptive transmission from the orofacial region. In this study, c-fos expression indicated by Fos immunohistochemistry in the caudal spinal trigeminal nucleus induced by subcutaneous injection of formalin into the lateral face of the rat was used as a marker for nociceptive neurons. The study sought to determine whether Fos-positive neurons express nitric oxide synthase, glutamate N-methyl-D-aspartate type receptor subunit 1, and glutamate alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid type receptor subunit 2r3; and whether they project to the thalamus. After formalin injection, many Fos-positive nuclei appeared in the superficial laminae of the ipsilateral trigeminal nucleus. Confocal laser scanning microscope revealed that almost all neurons with Fos immunofluorescent nuclei were colocalised with N-methyl-D-aspartate receptor 1, 94% with glutamate receptor 2r3 and 14% with nitric oxide synthase. Some of them were closely related to neurons labelled by nitric oxide synthase. Lastly, some of the Fos-positive neurons were labelled by tetramethylrhodamine-dextran injected into the trigeminothalamic tract or the thalamic region. The results suggested that activation of N-methyl-D-aspartate receptor 1 and glutamate receptor 2r3 upon glutamate release in response to noxious stimulation to the orofacial region might mediate c-fos expression in neurons involved in nociception. The expression of Fos in the neurons could also be mediated by nitric oxide produced from the same, as well as neighbouring neurons, when nociceptive stimulation persisted. Fos-positive neurons in the spinal trigeminal nucleus may project to the thalamus, relaying orofacial nociception to the higher sensory centre. q 2000 Elsevier Science B.V. All rights reserved. Keywords: Nociception; Colocalisation; Immunohistochemistry
1. Introduction Though the caudal spinal trigeminal nucleus ŽcSTN. has been implicated in nociceptive transmission, the neuroactive chemicals and connections of its neurons have not been studied in detail. This is due mainly to the fact that the neurons involved could not be precisely localised for the above studies. Recent investigation, however, demonstrated that noxious stimulation of the tooth pulp, or facial skin or after tooth extraction w9,50,53x or subcutaneous injection of formalin into the lateral face w23x caused c-fos expression in the cSTN. This led to the conclusion that the Fos-positive neurons are probably involved in nociception. The c-fos expressing neurons are present predominantly in lamina I, outer part of lamina II ŽIIo. and laminae V–VI of the spinal cord. These correspond to the terminal fields of
)
Corresponding author. Fax: q65-7787643; e-mail: [email protected]
primary nociceptive afferent fibres and to the distribution of nociresponsive neurons identified by electrophysiological recording w5,24,47x. In the case of the cSTN, they are also located predominantly in the superficial laminae of the nucleus. Hence, Fos, the nuclear protein encoded by the early gene c-fos, as demonstrated by immunohistochemistry, is a suitable indicator for the presence of nociceptive neurons activated by formalin injection into the lateral face. The formalin test as a means for assessing pain from the hindpaw, of upper lip or vibrissal pad of animals has, in fact, been widely used w7,14,36,56x. Recently, glutamate receptors and nitric oxide ŽNO. have been implicated in nociception and pain processing. In particular, activation of N-methyl-D-aspartate ŽNMDA. receptor has been shown to mediate sensitisation phenomena in spinal neurons following peripheral insult w55x. Spinal delivery of NMDA also produces hyperalgesia, an augmented response to noxious stimulation w1,8x. Moreover, this spinal hyperalgesia, as in the tonic phase of the
0006-8993r00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 6 - 8 9 9 3 Ž 9 9 . 0 2 3 1 6 - 1
108
S.-K. Leong et al.r Brain Research 855 (2000) 107–115
formalin test, is blocked by intrathecal injection of NMDA antagonists w38x. Inhibition of spinal NO synthesis blocks NMDA-induced hyperalgesia w29,39x, and thermal hyperalgesia resulting from nerve injury w41x. It also produces antinociception in the rat injected with formalin at the paw w49x. In addition to NMDA receptor, other types of glutamate receptors, the metabotropic and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid ŽAMPA. receptors have also been implicated in nociception w35,57x. Although glutamate receptors and NO have been widely reported to play important roles in spinal pain processing, very little is known in the case of orofacial nociception which is transmitted via afferent fibres of trigeminal ganglion neurons to the cSTN. From here, information is conveyed to the thalamus w52x then to the cerebral cortex. The present study aimed first to investigate the distribution of neurons in the cSTN that expressed Fos protein in response to subcutaneous formalin injection into the lateral face of the rat. This was followed by studying whether Ži. these neurons express glutamate receptors or NO synthase ŽNOS.; Žii. they project to the thalamus. The retrograde axonal tracer, tetramethylrhodamine-dextran ŽTMR-D., was injected into the thalamus or trigeminothalamic tract to reveal the trigeminothalamic neurons. Our preliminary study indicated that of all the glutamate receptors, NMDA receptor subunit 1 ŽNMDAR1. and AMPA receptor subunit 2r3 ŽGluR2r3. showed the most intense immunostaining in the neurons of the cSTN and should therefore play the most prominent role in nociception. Subsequently, the investigation focused on the two receptors.
2. Materials and methods A total of 24 adult male Wistar rats weighing between 250 and 300 g were used in the present study. Animal treatments and maintenance confirmed to the guidelines of the International Association for the Study of Pain w58x. The animals were divided into three groups. Group 1 consisted of 10 rats that received subcutaneous injection of dilute formalin into the lateral face only. Group 2 consisted of 10 rats which received a combination of thalamic or trigeminothalamic tract injection of TMR-D and formalin stimulation. After TMR-D injection, the rats were allowed to survive for 5–7 days and were perfused transcardially at 2 h after formalin injection. There were four rats in Group 3; these received saline injection only. Since our preliminary study showed that NMDAR1 and GluR2r3 staining in the superficial laminae of the cSTN were the strongest among all the NMDA and AMPA receptors, NMDAR1 and GluR2r3 were used for the colocalisation study. 2.1. Thalamic or trigeminothalamic tract injection TMR-D Ž3000 MW, anionic, lysing fixable; Molecular Probe, Eugene, OR. was dissolved in 0.1 M citrate-NaOH
at pH 3.0 and made up to 10% solution w26x. Anaesthesia of the rats was achieved by i.p. injection of chloral hydrate Ž35 mgrkg b.wt... Usually, no more than 7 mgrkg b.wt. was needed to maintain anaesthesia. The anaesthetised rats were placed on a stereotaxic apparatus and TMR-D solution was injected through a 5 ml microsyringe ŽExmire, 33 gauge. inserted into the right thalamic region or right trigeminothalamic tract. Injection was made according to the coordinates in the atlas by Paxinos and Watson w46x. In six rats, a total of 0.4–0.8 ml TMR-D solution was injected into the thalamic region. In each rat, the solution was injected into four regions: paracentral thalamic nucleus, posterior thalamic nuclear group, ventral posterolateral thalamic nucleus and ventral posteromedial thalamic nucleus. In four rats, 0.5 ml TMR-D solution was delivered to the right trigeminothalamic tract. Each dose of TMR-D was delivered within an interval of 10 min, with the needle of the syringe left in place for another 10 min. 2.2. Lateral face injection Under chloral hydrate anaesthesia, 0.5 ml of 4% formalin in saline was injected subcutaneously into multiple sites of the left lateral face of anaesthetised rats with a 0.5-ml syringe. Formalin injections were delivered to all Group 1 normal rats and Group 2 rats that had earlier received TMR-D injection into the right thalamic region or trigeminothalamic tract. Group 3 control rats received subcutaneous injection of 0.5 ml of physiological saline in the same manner as those receiving formalin injection. 2.3. Perfusion and tissue sectioning All the rats were anaesthetised with an overdose of chloral hydrate and perfused with 100 ml of Ringer’s solution 2 h after formalin or saline injection into the lateral face, followed by 500 ml of 0.1 M phosphate-buffer ŽpH 7.4. containing 4% Žwrv. paraformaldehyde. The caudal medulla and cervical spinal cord was then removed from the obex to the C2 segment and post-fixed in the same fixative for 2 h. Then the tissue was soaked in 20% sucrose in 0.1 M phosphate buffer overnight. Frozen sections were cut at 30 mm thickness in a cryostat ŽJung Frigocut 2800E. at y158C, and collected in a series of one in four sections in wells filled with phosphate buffered saline and 0.3% Triton X-100 ŽPBS-TX, pH 7.4.. 2.4. Immunohistochemistry and Õisualization of labelling 2.4.1. Fos immuno-DAB reaction or immunofluorescence Frozen sections for the detection of Fos protein product only were processed for Fos immuno-DAB reaction. They were washed for 2 h in 0.1 M PBS containing 0.3% Triton X-100, then blocked with 5% normal rabbit serum in PBS for half an hour. Polyclonal sheep anti-Fos ŽChemicon
S.-K. Leong et al.r Brain Research 855 (2000) 107–115
International, USA. were then applied on the sections overnight at a dilution of 1:1000 in 0.1 M PBS, containing 0.1% Triton X-100. After incubation, the sections were washed in 0.1 M PBS and placed in biotinylated rabbit anti-sheep IgG Ž1:200; Vector. for 1 h and washed again in 0.1 M PBS, followed by treatment with avidin–biotin–peroxidase complex ŽVector. for 1 h, and finally washed three times in 0.1 M PBS. Fos-LI was visualized with incubation in a nickel diaminobenzidine solution activated by 0.005% hydrogen peroxide for 3–4 min. Sections were then mounted on clean gelatin-coated slides and air-dried. They were dehydrated and mounted with permount and covered with coverslips. For Fos immunofluorescence, the sections were washed in 0.1 M PBS, after incubation with polyclonal sheep anti-Fos overnight, placed in donkey antisheep fluorescein conjugated IgG ŽChemicon International, USA. at a dilution of 1:160 for 1 h, and finally washed three times in 0.1 M PBS. After Fos immunofluorescence,
109
sections from rats receiving TMR-D injection into the thalamus or trigeminothalamic tract were collected on gelatin-coated slides, mounted with DAKO fluorescent mounting medium and covered with coverslips. Sections for double immunolabelling were stained for NOS, GluR2r3 and NMDAR1 immunofluorescence later. 2.4.2. Double labelling of Fos with NOS, GluR2r 3 and NMDAR1 After staining for Fos immunofluorescence, the sections that would be further processed for NOS, NMDAR1 or GluR2r3 immunofluorescence were incubated with polyclonal rabbit anti-NOS ŽChemicon International, USA, 1:500., or polyclonal rabbit anti-NMDAR1 ŽChemicon International, USA, 1 mr1 ml., or polyclonal rabbit antiGluR2r3 ŽChemicon International, USA, 1 mr1 ml.. After incubation, they were washed in 0.1 M PBS, and placed in cyanine 3 conjugated donkey anti-rabbit IgG ŽChemicon
Fig. 1. Photomicrograph of Fos labelling in the caudal medulla of the rat receiving formalin injection into the left lateral face. Note that nuclei intensively labelled by Fos immunohistochemistry are mostly concentrated in lamina I and the outer part of lamina II ŽIIo. of the left caudal spinal trigeminal nucleus ŽcSTN. with a few of them also scattered in the deeper laminae. Scale bar s 200 mm. Fig. 2. Photomicrograph of Fos labelling in the caudal medulla of the rat receiving physiological saline injection into the left lateral face. Note that the number of positive nuclei and their staining intensity is much less than that in Fig. 1. Scale bar s 200 mm.
110
S.-K. Leong et al.r Brain Research 855 (2000) 107–115
S.-K. Leong et al.r Brain Research 855 (2000) 107–115
International, USA. at a dilution of 1:600 for 1 h, and finally washed three times in PBS. They were then collected on gelatin-coated slides, mounted with DAKO fluorescent mounting medium and covered with coverslips. Control sections were incubated without primary antibodies. 2.4.3. Double labelling of TMR-D with Fos or NOS Sections from rats receiving TMR-D injection into the thalamus or trigeminothalamic tract were processed for Fos or NOS immunofluorescence as mentioned previously. They were then collected on gelatin-coated slides, mounted with DAKO fluorescent mounting medium and covered with coverslips. 2.4.4. Visualization of double labelling with confocal scanning microscopy Sections were examined with a Carl Zeiss, LSM410 confocal laser scanning microscope. Each section was initially scanned with a 488 nm laser line, and an emission filter BP 510–525, for the detection of fluorescein; and with a 543-nm laser line, and an emission filter LP 570, for the detection of cyanine 3 or TMR-D. 2.4.5. Cell counting To study the distribution of Fos-positive neurons in the cSTN, Fos-labelled nuclei were counted in 10 sections per rat through the main part of the left cSTN. To obtain the percentage of Fos-positive neurons colocalised with NMDAR-1 and GluR2r3, NOS or TMR-D in the cSTN, the cells were counted from 12 sections per rat. Sections for double staining of Fos with NMDAR-1 or GluR2r3 were collected from the same four rats while those for Fos and NOS double labelling were collected from another four rats as the rabbit anti-NOS was not available until a later date.
111
3. Results 3.1. Distribution of Fos-positiÕe neurons in the cSTN Fos labelling was observed in neuronal nuclei. In 18 rats receiving formalin injection into the left lateral face, an abundance of labelled nuclei was distributed mainly in lamina I, and the outer part of lamina II ŽIIo.. Some of them were also seen in the deeper laminae of the cSTN ŽFig. 1. ipsilateral to the side of formalin injection Žleft.. Counted from four rats, the number of Fos-positive neurons throughout the laminae of the left cSTN in one section ranged from 50 to 135. Fos-positive neurons located in laminae I and IIo ranged from 36 to 109 per section. There were very few labelled Fos-positive neurons in the deeper laminae of the nucleus, and such were less intensely labelled. In rats receiving physiological saline injection, much fewer Fos-positive neurons were seen in laminae I and IIo of the cSTN ŽFig. 2. ipsilateral to the side of injection, compared to the formalin injected ones. These were much less intensely stained. 3.2. Colocalisation of Fos with other neuroreactiÕe chemicals in the cSTN In eight rats receiving only formalin injection into the left lateral face, double immunofluorescence was used to detect whether Fos-positive neurons would also express glutamate receptor subunits and NOS. The sections incubated without primary antibodies were not stained. Counted from four rats, almost all the Fos-positive neurons were NMDAR1-positive ŽFig. 3.. 1272 of 1274 Fos-positive neurons were NMDAR1-positive. Most Fos-positive neurons were GluR2r3-positive ŽFig. 4.. A total of 889 out of 943, i.e., about 94% of Fos-positive neurons, were GluR2r3-positive. Only 14% of Fos-positive neurons Ži.e.,
Fig. 3. Photomicrograph shows that Fos-positive neurons induced by formalin injection are colocalised with NMDAR1 immunofluorescence Žarrows. in the superficial laminae of the cSTN. Note that the Fos-positive nuclei Žgreen. are surrounded by NMDAR1-positive cytoplasm Žred.. Fig. 4. Photomicrograph shows that Fos-positive neurons induced by formalin injection are colocalised with GluR2r3 immunofluorescence Žarrowed. in the superficial laminae of the cSTN. Note that the Fos-positive nuclei Žgreen. are surrounded by GluR2r3-positive cytoplasm Žred.. Fig. 5. Photomicrograph showing double labelling of Fos and NOS in the left cSTN of a rat receiving formalin injection into the left lateral face. Note that neurons containing nuclei labelled by Fos Žgreen. are more superficially located than those labelled by NOS Žred.. Note also that two Fos-positive nuclei are surrounded by NOS-positive cytoplasm Žarrow and arrowhead.. The insert shows a magnified view of the neuron indicated by an arrowhead. Fig. 6. Three Fos-labelled nuclei Žgreen. in the superficial laminae of the cSTN are shown in this photomicrograph. Two of them Žarrowed. are closely related to NOS-positive neurons Žred.. Fig. 7. Photomicrograph showing an NOS-positive neuron Žarrowed. with two processes Žred. in the superficial laminae of cSTN, one of which extends to the close vicinity of a neuron bearing a Fos-positive nucleus Žgreen.. Fig. 8. A TMR-D filled neuron Žarrowed. Žcytoplasm in red. contains a Fos-positive nucleus Žgreen. together with a few Fos-positive nuclei in the superficial laminae of the left cSTN in a rat receiving formalin injection in the left lateral face 5 days after TMR-D injection into the right trigeminothalamic tract. Insert shows the higher magnification of the neuron indicated by an arrow.
112
S.-K. Leong et al.r Brain Research 855 (2000) 107–115
48 of 354. were NOS-positive ŽFig. 5.. However, some Fos-labelled nuclei were observed to lie close to NOSpositive neuronal cell bodies ŽFig. 6. or neurites ŽFig. 7. in lamina IIo. 3.3. TMR-D labelled trigeminothalamic neurons The injection of the tracer TMR-D was targeted at the right trigeminothalamic tract or thalamus and the injection sites were confirmed by examination of the sections of the brain through the injected plane. In six rats receiving right thalamic injection and four rats receiving right trigeminothalamic tract injection of TMR-D, many neurons in the left cSTN were labelled by TMR-D. There were more labelled neurons in the deeper laminae than in laminae I and II. More labelled neurons were detected in the cSTN after trigeminothalamic tract injection than after thalamic injection of TMR-D. After formalin stimulation, TMR-D injected rats showed colocalisation of TMR-D with some Fos-positive cells in laminae I and II of the cSTN ŽFig. 8.. Counted from two rats receiving trigeminothalamic tract injection, 169 neurons were labelled with TMR-D. Only six TMR-D labelled neurons were Fos-positive in laminae I and II of the cSTN in the two rats. None of them were NOS-positive.
4. Discussion The present study showed that many Fos-positive nuclei appeared in the superficial laminae of the cSTN within 2 h of subcutaneous formalin injection into the lateral face of the rat. Most Fos-positive neurons expressed glutamate NMDA receptor subtype 1 ŽNMDAR1. and AMPA receptor subtype, GluR2r3. Only 14% of Fos-positive neurons expressed NOS, but some Fos-positive cells were found in close apposition to NOS-positive neurites and cell bodies. Lastly, some of the Fos-positive neurons were labelled by the neuronal tracer, TMR-D, injected into the trigeminothalamic tract or thalamic region. 4.1. Fos expression in the cSTN The cSTN is an important component in the relay of sensory information to higher brain centres w28x. A large number of studies have demonstrated that primary afferent fibres from the orofacial region terminate within the STN w17,44,45x and that laminae I and II of the STN, which receive small-diameter trigeminal afferent fibres, are primarily responsible for relaying orofacial non-reflexive nociceptive information w4x. The detection of the proto-oncogene c-fos and its protein product Fos has been regarded as a valuable tool for pain research w24x. Though not all Fos expressing neurons could be regarded as nociceptive neurons w20x, there is enough evidence to show that subcutaneous injection of
formalin can evoke profound hyperalgesia w56x and that intrathecally administered c-fos antisense oligodeoxynucleotides to the rat prior to formalin injections dose dependently decreased both Fos immunoreactivity in the dorsal horn of the rat lumbar cord and all nociceptive measures in the tonic phase of the formalin test w23x. Thus, Fos can be considered a reliable indirect marker for nociceptive neurons. The test for pain induced by formalin injection into the rat paw has gained wide recognition. Subcutaneous injection of formalin into the orofacial region has also been generally accepted as a reliable method in the study of orofacial pain w7,14x. Our finding that Fos-positive nuclei were principally localised in laminae I and IIo of the cSTN after subcutaneous injection of formalin into the lateral face of the rat is consistent with other observations on the cSTN after formalin administration into the lateral face, lips or supra-ocular region w25,33,54,30,51x. This also corresponds to the distribution of Fos-positive neurons in the superficial laminae of the spinal cord after cutaneous formalin stimulation in the hindpaw w24,47,5x. In this study, we observed that the number of Fos-positive neurons induced in the cSTN by formalin injection into the lateral face was much more than that induced by saline injection. Taking all this together, it is reasonable to surmise that though not all the Fos expressing neurons in the superficial laminae of the cSTN could be considered nociceptive neurons, as Fos expression may be induced by other forms of stimuli w20x, the large majority of them should be so regarded. On the other hand, it is critical to recognise that the absence of c-fos expression cannot be taken to mean an absence of neural activity w12x. 4.2. Colocalisation of Fos and NMDAR1 and GluR2r 3 in the neurons of the cSTN Many endogenous substances have been implicated in the genesis of pain. In addition to neuropeptides like substance P and calcitonin gene-related peptide, which are released in the spinal cord in vivo upon noxious peripheral stimulation w16,13,42x, glutamate through its receptor of the NMDA type has also been implicated in the production of the hyperalgesic state w56x. Activation of NMDA receptors causes release of substance P in the spinal cord w37x. Antagonists of NMDA receptors inhibit wind-up and other manifestations of spinal hyperexcitability w19,48,55x. Additionally, glutamate receptor of the NMDA type contributes significantly to the long-term change in central nociceptive neurons that accompany prolonged noxious chemical stimulation w19,43x. In the case of non-NMDA Žincluding AMPA and kainate. receptors, they most likely mediate the short-term activation of second-order neurons by peripheral nociceptors as well as other sensory receptors. Antagonism of non-NMDA receptors blocks the response of second-order neurons to all peripheral stimuli w11x. Non-NMDA receptors also contribute to all stages of
S.-K. Leong et al.r Brain Research 855 (2000) 107–115
hyperalgesia induced by inflammation w43x. The present results suggest an involvement of both NMDA and AMPA receptors in the short-term processing of inputs from nociceptive afferents at the level of the cSTN 2 h after formalin injection. The fact that NMDAR1 and GluR2r3 were expressed in most Fos-positive neurons in the superficial laminae of the cSTN, suggested that activation of both receptors upon glutamate release in response to noxious stimulation might mediate c-fos expression in neurons involved in hyperalgesia. This is supported by several lines of evidence obtained from pharmacological experiments, using subcutaneous injection of chemical irritants such as formalin or carrageenin as the noxious stimulation. These showed that intrathecal or systemic administration of NMDA receptor antagonists reduced the number of Fos-positive spinal neurons w6,27x or the expression of c-fos mRNA w15x, compared to controls. There was also correlation in the time course of the appearance of nociceptive behavioural signs and c-fos mRNA labelling w15x. No similar time course correlation has been performed in the study of orofacial pain induced by formalin injection into the face region. 4.3. Fos expressing neurons and NOS-positiÕe neurons in the cSTN In addition to glutamate receptors, recent studies reported that NO, a novel neurotransmitter in the nervous system w3x synthesized by NOS, could also play a role in nociceptive processing and could be involved in the production of a hyperalgesic state w18,39,40x. Pretreatment with intrathecal doses of NO inhibitor, N v-nitro-L-arginine methyl ester ŽL-NAME., significantly reduced the licking behavior associated with the injection of formalin into the left hindpaw of the rat w49x. It also reduced Fos labelling in the spinal cord induced by noxious mechanical or chemical irritant stimulation w32,22,49x. These two observations pointed clearly to a role of NO in c-fos expression caused by noxious stimulation. Thus, the colocalisation of Fos and NOS in the same neurons in the superficial laminae of the cSTN in response to subcutaneous formalin injection into the lateral face should be expected. The results indicated that only 14% of Fos-positive cSTN neurons expressed NOS immunoreactivity while the majority of NOS-positive neurons were Fos-negative. While Fos expression indicates activation of neurons in response to nociceptive stimulation, the absence of it in the NOS-positive neurons may not indicate that these neurons are not activated as a previous study has shown that not all neurons express the gene when activated w12x. The observation that some Fos-positive cSTN neurons were seen in close apposition to NOS-positive neuronal cell bodies as well as neuronal profiles is mirrored by that of Lee et al. w31x in the dorsal horn of the lumbar cord. Since NO is a gaseous molecule and is able to diffuse
113
through cell membranes freely, this neighbouring relationship suggests that NO might modulate the function of Fos-positive neurons via a paracrine mode. NO can diffuse from neuronal cell bodies or dendritic or axonal processes of these cells to adjacent neurons. Hence, NO may act on Fos-positive neurons through both intracellular as well as extracellular pathways to activate guanylyl cyclase, which in turn activates the cGMP pathway that may ultimately induce Fos expression and further bring about nociception. In addition to modulating Fos expression induced by nociception, NO may also modulate other functions as colocalisation of Fos with NOS was found in many oxytocin and vasopressin synthesizing neurons of the rat hypothalamus following stress stimuli w21x. 4.4. Fos expressing neurons projecting to the thalamus Of the TMR-D labelled neurons in the superficial laminae of the cSTN following injection of TMR-D injection into the right trigeminothalamic tract or thalamus, some were Fos-positive. These were obviously thalamic projecting neurons. This observation is consistent with that of Li et al. w33,34x who showed Fos expression in the cSTN after subcutaneous injection of formalin into the lips of rats. Since almost all Fos-positive neurons in the cSTN were NMDAR1-positive as demonstrated in this study, it is clear that some Fos and NMDAR1 coexpressing neurons in the cSTN were neurons projecting to the thalamus. They may relay orofacial nociception to the higher sensory centre. That not many TMR-D labelled neurons were found in the cSTN in the present study could be partially accounted for by the fact that some of the nociceptive neurons could project to other nuclei in the brain. For example, it had been demonstrated that formalin injection into the perioral region of the rat induced Fos expression in the parabrachial nucleus w59x and that formalin induced Fos-positive neurons in the cSTN projected to the parabrachial nucleus w54,33x. Thus, in addition to the thalamus, other nuclei may play important roles in conveying nociception from the orofacial region to the higher sensory centre. Significantly, none of the NOS-positive neurons were colocalised with TMR-D labelled ones, indicating that these neurons did not project to the thalamus. This is consistent with the finding of Dohrn et al. w10x. It may, therefore, be concluded that NOS-positive neurons in the superficial laminae of the cSTN are not projecting neurons. They may play a role in the local circuit for nociception as suggested by Aimar et al. w2x that NO producing neurons modulate the release of sensory neuropeptides in the rat substantia gelatinosa of the dorsal horn.
Acknowledgements We wish to thank Professor Lawrence Kruger from the Department of Neurobiology, School of Medicine, UCLA
114
S.-K. Leong et al.r Brain Research 855 (2000) 107–115
for his valuable comments on the manuscript. This work was supported by grants RP940320 and RP960331 from the National University of Singapore. References w1x L.M. Aanonsen, G.L. Wilcox, Nociceptive action of excitatory amino acids in the mouse: effects of spinally administered opioids, phencyclidine and sigma agonists, J. Pharmacol. Exp. Ther. 243 Ž1987. 9–19. w2x P. Aimar, L. Pasti, G. Carmignoto, A. Merighil, Nitric oxide-producing islet cells modulate the release of sensory neuropeptides in the rat substantia gelatinosa, J. Neurosci. 18 Ž1998. 10375–10388. w3x D.S. Bredt, S.H. Snyder, Nitric oxide, a novel neuronal messenger, Neuron 8 Ž1992. 3–11. w4x J.G. Broton, J.W. Hu, B.J. Sessle, Effects of temporomandibular joint stimulation on nociceptive and nonnociceptive neurons of the cat’s trigeminal nucleus caudalis Žmedullary dorsal horn., J. Neurophysiol. 59 Ž1988. 575–1589. w5x E. Bullitt, Somatotopy of spinal nociceptive processing, J. Comp. Neurol. 312 Ž1991. 279–290. w6x V. Chapmann, P. Honore, J. Buritova, J.M. Besson, The contribution of NMDA receptor activation to spinal c-fos expression in a model of inflammatory pain, Br. J. Pharmacol. 116 Ž1995. 1628–1634. w7x P. Clavelou, J. Pajot, R. Dallel, P. Raboisson, Application of the formalin test to the study of orofacial pain in the rat, Neurosci. Lett. 103 Ž1989. 349–353. w8x T.J. Coderre, R. Melzack, The distribution of excitatory amino acids to central sensitization and persistent nociception after formalin-induced tissue injury, J. Neurosci. 12 Ž1992. 3665–3670. w9x F. Coimbra, A. Coimbra, Dental noxious input reaches the subnucleus caudalis of the trigeminal complex in the rat, as shown by c-fos expression upon thermal or mechanical stimulation, Neurosci. Lett. 173 Ž1994. 201–204. w10x C.S. Dohrn, M.A. Mullett, R.H. Price, A.J. Beitz, Distribution of nitric oxide synthase-immunoreactive interneurons in the spinal trigeminal nucleus, J. Comp. Neurol. 346 Ž1994. 449–460. w11x P.M. Dougherty, J. Palecek, V. Paleckova, L.S. Sorkin, W.D. Willis, The role of NMDA and non-NMDa excitatory amino acid receptors in the excitation of primate spinothalamic tract neurons by mechanical, chemical, thermal, and electrical stimulation, J. Neurosci. 12 Ž1992. 3014–3025. w12x M. Dragunow, R. Faull, The use of c-fos as a metabolic marker in neuronal pathway tracing, J. Neurosci. Methods 29 Ž1989. 261–265. w13x A.W. Duggan, I.A. Hendry, C.R. Morton, W.D. Hutchison, Z.Q. Zhao, Cutaneous stimuli releasing immunoreactive substance P in the dorsal horn of the cat, Brain Res. 451 Ž1988. 261–271. w14x E. Eisenberg, B.P. Vos, A.M. Strassman, The NMDA antagonist memantine blocks pain behavior in a rat model of formalin-induced facial pain, Pain 54 Ž1993. 301–307. w15x K.J. Elliott, M. Brodsky, A.D. Hynansky, K.M. Foley, C.E. Inturrisi, Dextromethorphan suppresses both formalin-induced nociceptive behavior and the formalin induced increase in spinal cord c-fos mRNA, Pain 61 Ž1995. 401–409. w16x V.L. Go, T.L. Taksh, Release of substance P from the cat spinal cord, J. Physiol. 391 Ž1987. 141–167. w17x F. Greenwood, An electrophysiological study of the central connections of primary afferent nerve fibres from the dental pulp in the cat, Arch. Oral Biol. 18 Ž1973. 771–785. w18x J.E. Haley, A.H. Dickenson, M. Schachter, Electrophysiological evidence for a role of nitric oxide in prolonged chemical nociception in the rat, Neuropharmacology 31 Ž1992. 251–258. w19x J.E. Haley, A. Sullivan, A. Dickenson, Evidence for spinal Nmethyl-D-aspartate receptor involvement in prolonged chemical nociception in the rat, Brain Res. 518 Ž1990. 218–226.
w20x J.A. Harris, Using c-fos as a neural marker of pain, Brain Res. Bull. 45 Ž1998. 1–8. w21x S. Hatakeyama, Y. Kawai, T. Ueyama, E. Senba, Nitric oxide synthase-containing magnocellular neurons of the rat hypothalamus synthesize oxytocin and vasopressin and express Fos following stress stimuli, J. Chem. Neuroanat. 11 Ž1996. 243–256. w22x P. Honore, V. Chapman, J. Buritova, J.M. Besson, Reduction of carrageenin oedema and the associated c-fos expression in the rat lumbar spinal cord by nitric oxide synthase inhibitor, Br. J. Pharmacol. 114 Ž1995. 77–84. w23x W.Y. Hou, B.C. Shyu, T.M. Chen, J.W. Lee, J.Y. Shieh, W.Z. Sun, Intrathecally administered c-fos antisense oligodeoxynucleotide decreases formalin-induced nociceptive behavior in adult rats, Eur. J. Pharmacol. 329 Ž1997. 17–26. w24x S.P. Hunt, A. Pini, G. Evan, Induction of c-fos-like protein in spinal cord neurons following sensory stimulation, Nature 328 Ž1987. 632–634. w25x K. Iwata, K. Kanda, Y. Tsuboi, K. Kitajima, R. Sumino, Fos induction in the medullary dorsal horn and C1 segment of the spinal cord by acute inflammation in aged rats, Brain Res. 678 Ž1995. 127–139. w26x T. Kaneko, K. Saeki, T. Lee, N. Mizuno, Improved retrograde axonal transport and subsequent visualization of tetramethylrhodamine ŽTMR.-dextran amine by means of an acidic injection vehicle and antibodies against TMR, J. Neurosci. Methods 65 Ž1996. 157–165. w27x L.J. Kehl, K.R. Gogas, L. Lichtblau, C.H. Pollock, M. Mayes, A.I. Basbaum, G.L. Wilcox, The NMDA antagonist MK801 reduces noxious stimulus induced FOS expression in the spinal cord dorsal horn, in: M.R. Bond, J.E. Charlton, C.J. Woolf ŽEds.., Proceedings of the 6th World Congress on Pain, Elsevier, New York, 1991, pp. 307–331. w28x J.P. Kelly, Trigeminal system, in: E.R. Kandel, J.H. Schwartz ŽEds.., Principles of Neural Science, Elsevier, New York, 1986, pp. 381–393. w29x K.F. Kitto, J.E. Haley, G.W. Wilcox, Involvement of nitric oxide in spinally mediated hyperalgesia in the mouse, Neurosci. Lett. 148 Ž1992. 1–5. w30x A. Laudanna, M.I. Nogueira, M. Mariano, Expression of Fos protein in the rat central nervous system in response to noxious stimulation: effects of chronic inflammation of the superior cervical ganglion, Braz. J. Med. Biol. Res. 31 Ž1998. 847–850. w31x J.H. Lee, R.H. Price, F.G. Williams, B. Mayer, A.J. Beitz, Nitric oxide synthase is found in some spinothalamic neurons and neurons processes that appose spinal neurons that express Fos induced by noxious stimulation, Brain Res. 608 Ž1993. 324–333. w32x J.H. Lee, G.L. Wilcox, A.J. Beitz, Nitric oxide mediates Fos expression in the spinal cord induced by mechanical noxious stimulation, Neurol. Report 3 Ž1992. 841–844. w33x J.L. Li, T. Kaneko, S. Nomura, Y.Q. Li, N. Mizuno, Association of serotonin-like immunoreactive axon with nociceptive projection neurons in the caudal spinal trigeminal nucleus of the rat, J. Comp. Neurol. 384 Ž1997. 127–141. w34x J.L. Li, Y.Q. Li, T. Kaneko, N. Mizuno, Preprodynorphin-like immunoreactivity in medullary dorsal horn neurons projecting to the thalamic regions in the rat, Neurosci. Lett. 264 Ž1999. 13–16. w35x S. Maione, I. Marabese, J. Leyva, E. Palazzo, V. de Novellis, F. Rossi, Characterisation of mGluRs which modulate nociception in the PAG of the mouse, Neuropharmacology 37 Ž1998. 1475–1483. w36x Y. Mineta, E. Eisenberg, A.M. Strassman, Distribution of Fos-like immunoreactivity in the caudal medullary reticular formation following noxious facial stimulation in the rat, Exp. Brain Res. 107 Ž1995. 34–38. w37x H.T. Liu, P.W. Mantyh, A.I. Basbaum, NMDA-receptor regulation of substance P release from primary afferent nociceptors, Nature 386 Ž1997. 721–724. w38x A.B. Malmberg, T.L. Yaksh, Hyperalgesia mediated by spinal gluta-
S.-K. Leong et al.r Brain Research 855 (2000) 107–115
w39x
w40x w41x
w42x
w43x
w44x w45x
w46x w47x
w48x w49x
mate or SP receptor blocked by spinal cyclooxygenase inhibition, Science 247 Ž1992. 1276–1279. A.B. Malmberg, T.L. Yaksh, Spinal nitric oxide synthesis inhibition blocks NMDA-induced thermal hyperalgesia and produced antinociception in the formalin test in rats, Pain 54 Ž1993. 291–300. S.T. Meller, G.F. Gebhart, Nitric oxide ŽNO. and nociceptive processing in the spinal cord, Pain 52 Ž1993. 127–136. S.T. Meller, P.S. Pechman, G.F. Gebhart, T.J. Maves, Nitric oxide mediates the thermal hyperalgesia produced in a model of neuropathic pain in the rat, Neuroscience 50 Ž1992. 7–10. C.R. Morton, W.D. Hutchison, Release of sensory neuropeptides in the spinal cord: study with calcitonin gene-related peptide and galanin, Neuroscience 31 Ž1989. 807–815. V. Neugebauer, T. Lucke, H.G. Schaible, N-methyl-D-aspartate ŽNMDA. and non-NMDA receptor antagonists block the hyperexcitability of dorsal horn neurons during development of acute arthritis in rat’s knee joint, J. Neurophysiol. 70 Ž1993. 1365–1377. S.G. Nord, Responses of neurons in rostral and caudal trigeminal nuclei to tooth pulp stimulation, Brain Res. Bull. 1 Ž1976. 489–492. W.M. Panneton, H. Burton, Corneal and periocular representation within the trigeminal sensory complex in the cat studied with transganglionic transport of horseradish peroxidase, J. Comp. Neurol. 199 Ž1981. 327–344. G. Paxinos, C. Watson, The Rat Brain in Stereotaxic Coordinates, 3rd edn., Academic Press, San Diego, 1996. R.W. Presley, D. Menetrey, J.D. Levine, A.I. Basbaum, Systemic ´ morphine suppresses noxious stimulus-evolked fos protein-like immunoreactivity in the rat spinal cord, J. Neurosci. 10 Ž1990. 323–335. K. Ren, Wind-up and the NMDA receptor: from animal studies to humans, Pain 59 Ž1994. 157–158. A.K. Rocher, M. Cook, G.L. Wilcox, K.C.A. Kajander, A nitric oxide synthesis inhibitor ŽL-NAME. reduces licking behavior and Fos-labelling in the spinal cord of rats during formalin-induced inflammation, Pain 66 Ž1996. 331–341.
115
w50x A.M. Strassman, B.P. Vos, Y. Mineta, S. Naderi, D. Borsook, R. Burstein, Fos-like immunoreactivity in the superficial medullary dorsal horn induced by noxious and innocuous thermal stimulation of facial skin in the rat, J. Neurophysiol. 70 Ž1993. 1811–1821. w51x R. Terayama, N. Nagamatsu, T. Ikeda, T. Nakamura, O.I. Rahman, S. Sakoda, R. Shiba, T. Nishimori, Differential expression of Fos protein after transection of the rat infraorbital nerve in the trigeminal nucleus caudalis, Brain Res. 768 Ž1997. 135–146. w52x P.M.E. Waite, D.J. Tracey, Trigeminal sensory system, in: G. Paxinos ŽEd.., The Rat Nervous System, Academic Press, New York, 1995, pp. 705–724. w53x S. Wakisaka, Y. Sasaki, H. Ichikawa, S. Matsuo, Increase in c-foslike immunoreactivity in the trigeminal nucleus complex after dental treatment, Proc. Finn. Dent. Soc. 88 Ž1992. 551–555. w54x L.G. Wang, H.M. Li, J.S. Li, Formalin induced FOS-like immunoreactive neurons in the trigeminal spinal caudal subnucleus project to contralateral parabrachial nucleus in the rat, Brain Res. 649 Ž1994. 62–70. w55x C.J. Woolf, S.W.N. Thompson, The induction and maintenance of central sensitization is dependent on N-methyl-D-aspartic acid receptor activation: implications for the treatment of post-injury pain hypersensitivity states, Pain 44 Ž1991. 293–300. w56x T.L. Yaksh, A.B. Malmberg, Central pharmacology of nociceptive transmission, in: P.D. Wall, R. Melzack ŽEds.., The Textbook of Pain, Churchill-Livingstone, Edinburgh, 1994, pp. 165–200. w57x P.K. Zahn, E. Umali, T.J. Brennan, Intrathecal non-NMDA excitatory amino acid receptor antagonists inhibit pain behaviors in a rat model of postoperative pain, Pain 74 Ž1998. 213–223. w58x M. Zimmermann, Ethical guidelines for investigation of experimental pain in conscious animals, Pain 16 Ž1983. 109–110. w59x O. Hermanson, A. Blomqvist, Subnuclear localization of FOS-like immunoreactivity in the parabrachial nucleus after orofacial nociceptive stimulation of the awake rat, J. Comp. Neurol. 387 Ž1997. 114–123.