- Email: [email protected]
c-FOS-like immunoreactivity in rat brainstem neurons following noxious chemical stimulation of the nasal mucosa
Neuroscience Vol. 41, No. 213, pp. 629-641, Printed in Great Britain
0306-4522/91
1991
$3.00 + 0.00
Pergamon Press plc 0 1991 IBRO
c-FOS-LIKE IMMUNOREACTIVITY IN RAT BRAINSTEM NEURONS FOLLOWING NOXIOUS CHEMICAL STIMULATION OF THE NASAL MUCOSA F. ANTON,*? T. HERDEGEN,~P. PEPPEL~ and J. D. LEAH$ tbrstitut fur Physiologie und Biokybernetik, Universitltsstr. 17, D-8520 Erlangen, F.R.G. $11. Physiologisches Institut, Im Neuenheimer Feld 326, D-6900 Heidelberg, F.R.G. Abstract-It has previously been shown that noxious and non-noxious peripheral stimuli induce c-fos expression in spinal dorsal horn neurons. In the present study we have examined the expression of c-fos in brainstem neurons following noxious chemical stimulation of the respiratory region of the nasal mucosa. In urethane-anaesthetized rats we injected mustard oil or applied CO, pulses to the right nasal cavity. In control animals we applied paraffin oil or a continous flow of air. A further group of control animals was anaesthetized and not subjected to any experimental treatment. Two hours after the first stimulus the rats were perfused with 4% phosphate-buffered paraformaldehyde. Brainstem sections were incubated with primary antiserum against the FOS protein and processed according to the ABC method. Only the mustard oil-treated rats had obvious signs of rhinitis and displayed FOS-positive cells in laminae I and II of the subnucleus caudalis and in the subnucleus interpolaris of the trigeminal brainstem nuclear complex as well as in the medullary lateral reticular nucleus. These areas are known to be involved in the processing of nociceptive information. Although CO, pulses applied to the nasal mucosa are known to evoke pain sensations in man we did not observe any FOS-positive neurons in trigeminal and reticular brainstem areas of CO,-treated rats. This lack of c-fos expression probably results from the fact that unlike mustard oil, CO, did not induce any apparent inflammatory reactions. In all animals c-fos expression was found in the nucleus of the solitary tract and in the area postrema. Staining in these areas might partly result from factors related to anaesthesia, changed respiration parameters and stress. Since the mustard oil-treated rats displayed the highest levels of immunoreactivity in the nucleus of the solitary tract and in the area postrema, additional effects specifically related to nociceptive input are very likely.
The ethmoidal nerve innervates the respiratory region of the nasal mucosa and is believed to constitute the afferent limb of protective upper airway reflexes. Within the trigeminal brainstem nuclear complex (TBNC) ethmoidal primary afferents have been shown in cat and rat to project exclusively to laminae I and II of subnucleus caudalis (SNC) and to portions of subnucleus interpolaris (SNI),‘,*’ areas which are known to contain numerous nociceptive neurons.9*‘5 Peripheral and central electrophysiological recordings in cat5*28,2g provide further support for the nociceptive function of the ethmoidal nerve. In rat, additional ethmoidal primary afferent terminals have been localized in the interstitial subnucleus of the nucleus tractus solitarius (NTS),* an area known to be involved in control of the respiratory rhythm.i4 Psychophysical investigations in humans have shown that noxious chemical stimulation of the nasal *To whom correspondence should be addressed. ABC, avidin-biotin complex; AP, area postrema; FOS-LI, FOS-like immunoreactivity; LRN, lateral reticular nucleus; NGS, normal goat serum; NTS, nucleus tractus solitarius; PB, phosphate buffer; PBS, phosphate-buffered saline; PBST, PBS with 0.2% Triton X- 100; SNC, subnucleus caudalis; SNI, subnucleus interpolaris; TBNC, trigeminal brainstem nuclear complex.
Abbreviations:
629
mucosa with capsaicin or CO, induces pain sensations and evokes protective reflexes like secretion and sneezing.‘2*22~23 The goal of the present study was to characterize this system further by mapping second and higher order brainstem neurons activated by noxious chemical stimulation of the nasal mucosa. A recently developed method to perform this mapping at the single cell level is based on the expression of the proto-oncogene c-fos which is triggered by extrinsic stimuli. The FOS protein is a nuclear protein participating in the control of the transcription rate of target genes which can play a role in long term alterations of cellular function.36 In spinal cord neurons FOS is induced by sensory stimulation.‘* More specifically, in superficial laminae of the dorsal horn, FOS rapidly appears following the application of noxious heat or chemical stimuli.‘8*25 Starting from the hypothesis that the ethmoidal nerve mainly carries nociceptive information we have examined the central projections of this nerve in the companion paper.* In the present study we used FOS-like immunoreactivity (FOS-LI) to map the distribution of brainstem neurons45 activated by noxious chemical stimulation of the nasal mucosa. These results have been partly described in a short communication.’
630
F. ANTON VI ul EXPERIMENTAL
PROCEDURES
Stimulating procedures
Twenty male Wistar rats weighing 320440 g were lightly anaesthetized with urethane (1 g/kg, i.p.) and divided into five experimental groups, according to the applied stimulation procedure. Mustard oil group (n = 6). In order to stimulate the ethmoidal nociceptors 10~1 of 5% mustard oil (allyl-isothiocyanate) in paraffin oil were injected into the nasal meatus. A blunt Hamilton syringe was used to apply liquid chemicals to the right nasal cavity (for further details see companion paper).? Control group 1 (n = 2). To control for non-specific effects related to the injection procedure, 10 pl of pure paraffin oil were injected into the nasal cavity. CO, group (n = 6). Noxious stimulation of the nasal mucosa was done with 100% CO,. To prevent long lasting hyperventilation we applied CO, pulses of 15 s duration followed by interstimulus intervals of 15 s rather than continuous flow of the stimulating gas. These pulses were presented in a humidity-saturated continuous airflow (20ml/s) adjusted to a temperature of 28°C. The whole stimulating procedure lasted 1 h. For gas application to the nasal mucosa a thin plastic tube was inserted into the right nasal cavity. Gas leaks were prevented by adjusting the tube’s diameter individually to seal the nostril. This tube was connected to an olfactometerz2.*r presenting a continuous airflow in which defined stimulating gas pulses can be applied without any changes in humidity, temperature and total flow rate. Control group 2 (n = 2). To control for non-specific effects related to the stimulating procedure described in the CO, group we presented a continuous similar airflow without any CO, application. Confrol group 3 (n = 4). To control for background FOS-LI and/or effects related to anaesthesia in certain brainstem areas, four animals were anaesthetized 2 h prior to the perfusion and did not undergo any further treatment. Tissue processing
Two hours after the initiation of the stimulation procedures the rats were deeply anaesthetized with thiobutabarbital sodium (Inactin”, 125 mg/kg, i.p.) and perfused through the aorta with 7&100 ml warm phosphate-buffered saline (PBS) containing 0.1% sodium nitrite and 10,000 units/l heparin followed by 50 ml of warm fixative (4% paraformaldehyde in PBS). The final cold flush consisted of 200 ml of the same fixative. The brains were removed and postfixed for &6 h in cold fixative containing 30% sucrose. They were then stored in a 30% sucrosephosphate buffer (PB: 0.1 M, pH 7.4) at 4°C for 3-8 days. Serial coronal brainstem sections (40 pm) were cut on a freezing microtome and processed for immunohistochemistry ai free floating sections. We used chilled uhosphate buffered saline with 0.2% Triton X- 100 (PBST)’ as diluent for all immunoreagents and rinsing solutions. The sections were first rinsed in PBST and then incubated in a blocking solution containing 1.5% normal goat serum (NGS) at 37°C for 1 h. The tissue was then incubated for 36 h in polyclonal rabbit antiserum directed against the c-fos protein (Medac, Hamburg), diluted 1: 1000. After that the tissue was processed using the avidin-biotin complex (ABC) method.” Following a final rinse the sections were developed in a solution of 0.01% diaminobenzidine and 0.02% hydrogen peroxide. The staining procedure was stopped by washing the tissue in doubly distilled water. The sections were then mounted on chrome-alum-gelatine coated slides, air dried and coverslipped. The brainstem sections were examined under a light microscope. The location of immunoreactive cell bodies was mapped with camera lucida drawings and microphoto-
graphs were taken. From each above, the rat with the highest chosen to screen the number of several distinct brainstem areas. were made by using a stereotaxic
of the groups described density of labelling WEiS c-fos-positive neurons in Anatomical correlations atlas.4’
RESULTS
The reactions of the animals to the stimulation procedures and the location of FOS-positive brainstem cells within the different groups exhibited the following features. Mustard
oil group
Immediately following mustard oil application all rats strongly expired through the nose and then stopped breathing for up to 15 s. Signs of inflammation like mucosal swelling, reddening and secretion could rapidly be observed and were still present when the animals were about to be perfused. As shown in camera lucida drawings of coronal sections through the brainstem (Fig. l), mustard oil treatment caused extensive FOS-LI in several brainstem nuclei. In the TBNC c-fos-positive cells could only be observed in the ipsilateral SNC and SNI. Labelling started about 0.7 mm caudal to the obex at the rostra1 pole of SNC and extended for a distance of about 1.4 mm in the rostra1 direction to the caudal pole of SNI. FOS-positive cell nuclei were strictly located in the superficial laminae (I and II) of the ventrolateral portion of SNC (see Figs 1 and 2a). In SNI the density of labelling decreased rapidly, stained nuclei tended to be situated more medially than in SNC and could also be observed in dorsal aspects of the subnucleus (Figs 1 and 2b). Counts across the ipsilateral SNC and SNI revealed up to 246 labelled cells (see Table 1). No signs of FOS-LI could be found in the subnucleus oralis or in the main sensory nucleus. c-fos-Positive could be observed neurons bilaterally in the NTS, as shown in Figs 1 and 3. Labelling started about 0.2 mm caudal to the obex and spanned a distance of about 1.8 mm in the rostra1 direction. Up to 402 stained cells were bilaterally counted across this nucleus, as shown in Table 1. A large number of stained cell nuclei (up to 225) could also be observed throughout the area postrema (AP) (see Figs I and 4). Finally, the mustard oil-treated rats displayed bilateral FOS-LI (up to 125 labelled cells, see Table 1) in the medullary lateral reticular nucleus (LRN), as shown in Figs 1 and 5. Control
group
Following apnoea or animals. We the trigeminal control rats.
1 the paraffin oil injection no signs of inflammation were observed in the could not detect any FOS-LI in any of or reticular brainstem nuclei of these Only NTS and AP displayed a rather
c-fos after noxious stimulation of the nasal mucosa
631
Fig. 1. Camera lucida drawings of coronal brainstem sections from a mustard oil-treated rat showing localization of FOS-positive neurons (dots) in different nuclei along the rostrocaudal axis. The dashed lines indicate subnuclei of the TBNC. (1) FOS-LI in SNC (a), in the transition zone between SNC and SNI (b) and in SNI (c). (2) FOS-positive cells in NTS, (3) in AP and (4) in LRN. PD, pyramidal decussation; ‘IT, trigeminal tract. Numbers to the right of each figure indicate the distance in millimetres rostra1 (+) or caudal (-) to the obex. low density of labelled cell nuclei (up to 48 and 72 cells respectively, see Table 1).
co,
group
The six rats stimulated with CO2 sneezed and decreased their respiration frequency following the presentation of the first CO2 stimuli. These reactions lasted 1-2 min and vanished during the course of the remaining stimulation procedure. No obvious signs of inflammation could be observed. In these animals the TBNC was devoid of FOS-LI. In addition no FOS-positive cells could be found in the reticular formation. In the caudal NTS bilateral labelling of cell nuclei (up to 195 cells) could be observed. The labelling was consistently less dense than in the mustard oil group. As displayed in Table 1, up to 130 FOS-positive cells were counted in AP, again considerably less than in the mustard oil group. Control group 2
In the two rats in which a continuous tIow of air was applied to the nasal mucosa we observed a slight twitching of the nose and face muscles which lasted some seconds following the start of the airflow presentation. In these control animals we did not find any FOS-positive neurons in TBNC and in the reticular formation. In NTS we observed up to 93 labeled nuclei, whereas up to 70 stained neurons were counted in AP.
Control group 3
The four rats which were anaesthetized and not subjected to any further treatment displayed a rather low density of FOS-LI in NTS (up to 65 cells, bilaterally) and in AP (up to 46 cells). No signs of labelling were observed in any of the other brainstem nuclei mentioned above (see Table 1).
DISCUSSION
FOS-like immunoreactivity in trigeminal brainstem nuclear complex and lateral reticular nucleus
In this study two different noxious chemical stimuli, mustard oil and CO, were applied to the nasal mucosa of anaesthetized rats. In rat, mustard oil has been shown to excite polymodal C-nociceptors and to cause neurogenic inflammation which in turn leads to sustained firing for up to 30 min and to enhanced thermal responsiveness of these units.43 In humans, topical application of mustard oil to the skin leads to a burning pain sensation and to local inflammatory responses which persist for several minutes following washout of the application site.” CO2 pulses applied to the nasal mucosa have been reported to induce pain in man.22*23In a rat skinsaphenous nerve in vitro preparation, electrophysiological recordings revealed that CO2 specifically
F. ANTON et al.
Fig. 2a.
634
F. Awrorj
excites nociceptive primary afferents and that this effect can probably be attributed to lowering of the intracellular pH to acid levels.48 In these experiments the nociceptors displayed an ongoing activity as long as CO, was applied to the receptive fields, Human
et ai.
psychophysical experiments in our laboratory revealed, however, that long lasting CO, pulses (up to 20 s) applied to the nasal mucosa induce very phasic pain sensations never lasting more than 34 s (unpubIished observations). Under these conditions the
Fig.
?a
c-fos after noxious stimulation of the nasal mucosa
Fig. 3b. Fig. 3. Photographs of coronal sections displaying immunoreactive cell nuclei in caudal (a) and rostra1 (b) NTS of a mustard oil-treated rat. x 119. Boxes in the schematic drawings indicate areas shown in the photomicrographs.
635
635
Fig. 4. Fh~t~mi~r~~a~h displa$ng RX-positive cells in AP following mustard oif treatment. x 119. The area shown in the ptrotograph is indicated by the box in the schematic drawing.
secretion of mucus induced by noxious stimulation of the nasal mucosa probably results in fast buffering of CO, related pW changes.
In the present study the animals treated with mustard oil displayed c-fos-positive cells in the ~rigern~n~l SNC and SNI as well as in the lateral
c-fos after noxious stimulation of the nasal mucosa
Fig. 5. Photograph of a coronal section showing FOS-LI in LRN of a mustard oil-treated rat. x 119. The box in the schematic drawing indicates the photographed area.
637
F. ANTON et al.
638 Table
1. Numbers
of immunoreactive brainstem nuclei Brainstem
Experimental group Mustard Oil Control 1 co, Control 2 Control 3
cells
in different
nuclei
TBNC
LRN
NTS
AP
246
12s
402 48 195 93 65
22s 72 130 70 46
-
Counts from the rat displaying labelling in each experimental
the highest density group are shown.
of
reticular nucleus (LRN), whereas in the CO,-treated rats these nuclei were devoid of labelling. This discrepancy can probably be explained by the differences of the physiological reactions evoked by mustard oil and CO* application. The mustard oil-treated rats showed obvious signs of inflammation, like mucosal swelling, reddening and secretion. As discussed above, this neurogenic inflammation probably caused an ongoing activity in ethmoidal nociceptive primary afferents and resulted in a high degree of synaptic input in higher order neurons. In contrast, the CO, pulses used in our study probably induced rather phasic discharges of the ethmoidal nociceptive primary afferents. In addition no signs of inflammation could be observed in these animals. It is thus very likely that the summation of synaptic input to the respective higher order neurons was low compared to the mustard oil condition. Since the amount of FOS protein produced is probably directly related to the degree of synaptic activation5’ we conclude that the lack of FOSpositive neurons in the TBNC and LRN of CO,treated animals is likely to be due to comparatively low activity levels of central neurons under these conditions. Although the function of the FOS protein is not yet known, it is believed to participate in long lasting functional alterations of neurons.‘8s25 Such long term changes of neuronal function could be particularly relevant in tonic, inflammatory types of pain which can, for example, trigger persistent central mechanisms of sensitization.‘633853 Since none of the control groups displayed any FOS-LI in TBNC or LRN we can conclude (1) that nasal mucosa stimuli which do not evoke noxious input or only lead to brief, phasic discharges of nociceptive primary afferents do not induce c-fos expression in the trigeminal system or in the lateral reticular formation and (2) that no background labelling related to our anaesthetic conditions can be observed in these brainstem areas (concerning depressing effects of anaesthesia see Basbaum et ~f.).~ Within TBNC the localization of c-fos-positive cells corresponded exactly to the location of the ethmoidal primary afferent terminals.’ Thus it is very likely that the majority of stained cells were driven by monosynaptic ethmoidal input. The lack of a significant population of polysynaptically excited cells in
areas not receiving primary afferent input could be related to anaesthesia.4 However, since our rats were lightly anaesthetized and still showed pronounced nociceptive reflexes we probably would not have missed a whole population of polysynaptically activated cells. One major function of the TBNC areas containing FOS-LI is the processing of nociceptive information (see companion paper).2 Spinal dorsal horn and SNC lamina I neurons project to many brainstem and diencephalic nuclei including dorsal and ventral areas of the reticular formation,35a46 nucleus tractus solitarius,32 parabrachial area,‘9~s0hypothalamus’ and to medial and ventrolateral thalamic nuclei (for review see Willis).‘* Projections of nociceptive SNI neurons have not been examined as extensively. To gain further insight into functional aspects it would be interesting to know whether all of these areas, or preferentially some, receive input from highly activated trigeminal nociceptive neurons. Menetrey et ~1.~’performed a double labelling study at the level of the spinal dorsal horn and found a significant portion of FOS-positive neurons projecting to the areas mentioned above. Differential injections of retrograde tracer into single nuclei of the brain will increase our knowledge about projections and functions of FOS-positive nociceptive brainstem neurons. Recent reports indicate that inflammatory reactions which lead to sensitized responses of nociceptive primary afferents and hence to significant peptide release from their central terminalsi might be required to induce c-fos expression in the superficial spinal dorsal horn. Intrathecal application or superfusion of the spinal cord with neuropeptides like substance P, neurokinin A or bombesin caused c-fos staining in superficial laminae of the dorsal horn.25a5’ Williams et al.” reported that, independent of the nature of the cutaneous noxious stimulation, similar levels of c-fos immunoreactivity in the spinal dorsal horn are obtained if the stimuli lead to comparable intensities of inflammation as expressed by plasma extravasation and oedema. Furthermore, the pattern of FOS-LI in the superficial dorsal horn is coincident with the distribution of neurons that exhibit increased preproenkephalin or preprodynorphin mRNA levels in rat models of Moreover in arthritic peripheral inflammation. 20,40,44 rats, Weihe and co-workers49 have shown extensive co-localization of proenkephalin or prodynorphin opioids and FOS protein in the dorsal horn. The fact that FOS could regulate the preproenkephalin and preprodynorphin genes under these conditions (see review paper by Morgan and Curran) stresses the importance of this nuclear protein in long term CNS processing of nociceptive input from inflamed tissue. In the present study the mustard oil-treated rats were the only ones displaying FOS-LI in the medullary LRN. This nucleus has been reported to receive input from spinal second order neurons via
c-fos
after noxious stimulation of the nasal mucosa
the lateral spinoreticular tract.35 The induction of in this nucleus thus resulted from polysynaptic activation of the respective neurons, which is a further indication that the urethane anaesthesia used in our study did not generally prevent c-fos expression in cells with polysynaptic input. LRN is known to be involved in sensorimotor integration because it is highly interconnected with structures like motor cortex, cerebellum, red nucleus, ventral and dorsal horns of the spinal cord and other structures.6~24~26~3’~34~35~42 The labelling observed in our study could thus be related to persistent modulation of motor reactions like facilitation of protective reflexes triggered by tonic noxious input. Concerning processing of nociceptive information, the LRN has more recently been reported to be involved in descending inhibition.“*‘3,37s47In rat, Janss and Gebhart*’ have proposed that spinal alpha, and serotonin receptors mediate the inhibition of nociceptive responses from the LRN. Alternatively to the modulation of motor reactions discussed above, the c-fos-positive LRN neurons observed in our study could thus reflect sustained descending inhibition of nociceptive input. At the moment we do not have any explanation for the fact that none of the nuclei in the rostra1 ventral medulla (see Basbaum and Fields),3 involved in the descending control of spinal nociceptive processing, displayed FOS-LI.
c-fos
C-fos like immunoreactivity in nucleus tractus solitarius and area postrema
The NTS is roughly divided into a medial and a lateral region. The medial part mainly receives visceral afferent input and is involved in integration of respiratory and cardiovascular parameters.’ The lateral part predominantly receives gustatory input.r4 The AP constitutes the caudal ventral floor of the fourth ventricle and contains chemosensitive neurons involved in respiratory contro13’ The animals of all five groups described above displayed at least a moderate amount of FOS-LI in these areas. The lowest numbers of labelled cells were found in the control groups. This finding could reflect a low background expression of the c-fos gene. Other authors have described scattered FOS-positive brainstem neurons in non-stimulated animals.45 Since the FOS-positive cells in our study were restricted to
639
NTS and AP it is more likely that effects related to anaesthesia, like changes in pulse rate, respiration frequency and volume, were involved. The highest densities of FOS-LI in NTS and AP were observed in the rats treated with noxious chemical stimuli, and the mustard oil-treated animals displayed a higher density than the CO,-stimulated ones. In both groups we observed protective upper airway reflexes like apnoea, sneezing and gasping. The dramatic changes in respiratory and cardiovascular parameters resulting from the noxious stimultion could have induced a more pronounced c-fos expression than in the control groups. The inhalation of CO2 can lead to an acidosis which in turn causes hyperventilation. To limit c-fos expression induced by these respiratory changes we did not apply the CO2 continuously but in the form of pulses. Consequently we did not see any signs of sustained hyperventilation during the CO2 stimulation. Since the mustard oil-treated rats were the only ones reacting with apnoea for a longer period, this factor could explain the highest density of labelling found in these animals. Concerning NTS, it is possible that the tonic noxious stimulation of ethmoidal primary afferents in the mustard oil group increased the amount of c-fos expression. In the interstitial subnucleus of NTS, c-fos expression could also have resulted from monosynaptic activation. This subnucleus, which is specifically involved in respiratory integration, has been shown to contain terminals of ethmoidal primary afferents (see Anton and Peppel).’ Menetrey and Basbaum3* showed presumably nociceptive spinal and medullary dorsal horn neurons to project to NTS and thus probably to be involved in somatovisceral and viscerovisceral reflex activation. Therefore the expression of c-fos is likely to have been induced by polysynaptic activation in a large population of NTS neurons. Finally, we cannot exclude that mustard oil as well as CO, could have reached branches of the cranial nerves IX and X via the pharynx, larynx and oral cavity (see Ref. 2). Since both substances cause taste sensations, gustatory induction of c-fos might have been possible in a population of NTS neurons. Acknowledgements-We would like to thank Mr K. Burian for excellent technical assistance. This work was supported by the DFG grant An 158/1-l.
REFERENCES
Anton F., Herdegen T., Peppel P. and Leah J. D. (1989) C-fos expression in rat brainstem neurones following noxious stimulation of the nasal mucosa. Eur. J. Neurosci. (Suppl.) 2, 184. Anton F. and Peppel P. (1991) Central projections of trigeminal primary a&rents innervating the nasal mucosa: a horseradish peroxidase study in the rat. Neuroscience 41, 617628. Basbaum A. I. and Fields H. L. (1984) Endogenous pain control systems: brainstem spinal pathways and endorphin circuitry. A. Rev. Neurosci. 7, 309-338. Basbaum A. I., Presley R., Menttrey D., Chi S. J. and Levine J. D. (1989) Somatic, articular and visceral noxious-stimulus-evoked expression of c-fos in the spinal cord of the rat: differential patterns of activity and modulation by analgesic agents. In Processing of Sensory Information in the Superficial Dorsal Horn of the Spinal Cord (eds Cervero F., Bennett G. J. and Headley P. M.), pp. 3655382. Plenum Press, New York.
640
F. ANTONet al.
5. Beuerman R. W. (1975) Neurons in trigeminal nucleus and reticular formation excited by ethmoidal nerve stimulation. Brain Res. 92, 479484. 6. Brodal P., Marsala J. and Brodal A. (1967) The cerebral cortical projection to the lateral reticular nucleus in the cat, with special reference to the sensorimotor cortical areas. Brain Res. 6, 252-274. 7. Burstein R., Cliffer K. D. and Giesler G. J., Jr (1987) Direct somatosensory projection from the spinal cord to the hypothalamus and telencephalon. J. Neurosci. 7, 4159-4164. 8. Bystrzycka E. K. and Nail B. S. (1985) Brainstem nuclei associated with respiratory, cardiovascular and other autonomic functions. In The Rat Neroous System (ed. Paxinos G.), Vol. 2, pp. 95-110. Academic Press, Sydney. 9. Dubner R. and Bennett G. J. (1983) Spinal and trigeminal mechanisms of nociception. A. Reu. Neurosci. 6, 381418.
10. Duggan A. W., Morton C. R., Hendry A. I. and Hut&son W. D. (1989) Peripheral stimuli releasing neuropeptides in the dorsal horn of the cat. In Processing of Sensory Information in the Superficial Dorsal Horn of the Spinal Cord (eds Cervero F., Bennett G. J. and Headley P. M.), pp. 347-364. Plenum Press, New York. 11. Gebhart G. F. and Ossinov M. (1986) Characterization of inhibition of the sninal nociceutive tail-flick reflex in the rat from the medullary lateral reticular nucleus. J. Neurosci. 6, 701-713. _ 12. Geppetti P., Fusco B. M., Marabini S., Maggi C. A., Fan&ha& M. and Sicuteri F. (1988) Secretion, pain and sneezing induced by the application of capsaicin to the nasal mucosa in man. Br. J. Pharmac. 93, 509-514. 13. Hall J. G., Duggan A. W., Morton C. R. and Johnson S. M. (1982) The localization of brainstem neurons tonically inhibiting dorsal horn neurons of the cat. Brain Res. 244, 215-222. 14. Hamilton R. B. and Norgren R. (1984) Sensory projections of gustatory nerves in the rat. J. camp. Neurol. 222, X%577. 15. Havashi H.. Sumino R. and Sessle B. J. (1984) Functional organization of trigeminal subnucleus interpolaris: no&eptive ‘and innocous afferent inputs, projections to thalamis, cerebellum, and spinal cord, and descending modulation from periaqueductal gray. J. Neurophysiol 51, 890-908. 16. Hoheisel U. and Mense S. (1989) Long-term changes in discharge behaviour of cat dorsal horn neurones following noxious stimulation of deep tissues. Pain 36, 239-249. 17. Hsu S. M., Raine L. and Fanger H. (1981) Use of avidin-biotin-peroxidase complex (ABC) in immunoperoxidase techniques. J. Histochem. Cyrochem. 29, 577-580. 18. Hunt S. P., Pini A. and Evan G. (1987) Induction of c-fos-like protein in spinal cord neurons following sensory stimulation. Nature 328, 632634. 19. Hylden J. L. K., Anton F. and Nahin R. L. (1989) Spinal lamina I projection neurons in the rat: collateral innervation of parabrachial area and thalamus. Neuroscience 28, 27-37. 20. Iadarola M. J., Douglass J., Civelli 0. and Naranjo J. R. (1988) Differential activation of spinal cord dynorphin and enkephalin neurons during hyperalgesia: evidence using cDNA hybridisation. Brain Res. 455, 205-212. 21. Janss A. J. and Gebhart G. F. (1987) Spinal monoaminergic receptors mediate the antinociception produced by glutamate in the medullary lateral reticular nucleus. J. Neurkci. 7, 2862-2873. 22. Kobal G. (1985) Pain-related electrical potentials of the human nasal mucosa elicited by chemical stimulation. Pain 22, 151-163. 23. Kobal G. and Hummel C. (1988) Cerebral chemosensory evoked potentials elicited by chemical stimulation of the olfactory and respiratory nasal mucosa. Electroenceph. clin. Neurophysiol. 71, 241-250. 24. Kiinzle H. and Wiesendanger M. (1974) Pyramidal connections to the lateral reticular nucleus in the cat: a degeneration study. Acta anat. 88, 105-114. 25. Leah J. D., Herdegen T. and Zimmermann M. (1989) Physiological and pharmacological induction of c-fos protein immunoreactivity in superficial dorsal horn neurons. In Processing of Sensory Information in the Superficial Dorsal Horn of the Spinal Cord (eds Cervero F., Bennett J. G. and Headley P. M.), pp. 307-310. Plenum Press, New York. 26. Lowey A. D., Wallach J. H. and McKellar S. (1981) Efferent connections of the ventral medulla oblongata in the rat. Brain Res. Rev. 3, 63-80.
27. Lucier G. E. and Egizii R. (1986) Central projections of the ethmoidal nerve of the cat as determined by the horseradish peroxidase tracer technique. J. camp. Neural. 247, 123-132. 28. Lucier G. E. and Egizii R. (1988) Characterization and projection of nociceptive input from the nasal cavity of the cat. In Proceedings of the Vth World Congress on Pain (eds Dubner R., Gebhart G. F. and Bond M. R.), pp. 541-547. Elsevier, Amsterdam. 29. Lucier G. E. and Egizii R. (1989) Characterization of cat nasal afferents and brainstem neurones receiving ethmoidal input. Expl Neurol. 103, 83-89. 30. Magerl W., GrImer G. and Handwerker H. 0. (1990) Sensations and local inflammatory responses induced by application of carbachol, dopamine, 5-HT, histamine and mustard oil to the skin in humans. Eur. J. Physiol. 415 (suppl. I), R107. 31 Marchand J. E. and Hagino N. (1983) Afferents to the periaqueductal gray in the rat: a horseradish peroxidase study. Neuroscience 9, 95-106.
32. Menetrey D. and Basbaum A. I. (1987) Spinal and trigeminal projections to the nucleus of the solitary tract: a possible substrate for somatovisceral and viscerovisceral reflex activation. J. camp. Neurol. 25S, 439450. 33. Menetrey D., Gannon A., Levine J. D. and Basbaum A. I. (1989) The expression of c-fos protein in presumed nociceptive interneurons and projection neurons of the rat spinal cord: anatomical mapping of the central effects of noxious somatic, articular and visceral stimulation. J. camp. Neurol. 285, 177-195. 34. Menetrey D., De Pommery J. and Besson J. M. (1984) Electrophysiological characteristics of lumbar spinal cord neurons backfired from lateral reticular nucleus in cat. J. Neurophysiol. 52, 595611. 35. Menetrey D., Roudier F. and Besson J. M. (1983) Spinal neurons reaching the lateral reticular nucleus, as studied in the rat by retrograde transport of horseradish peroxidase. J. camp. Neurol. 220, 439452. 36. Morgan J. E. and Curran T. (1989) Stimulus-transcription coupling in neurons: role of cellular immediate-early genes. Tren& Neurosci. 12(1l), 459462. 37. Morton C. R., Johnson S. M. and Duggan A. W. (1983) Lateral reticular regions and the descending control of dorsal horn neurons of the cat: selective inhibition by electrical stimulation. Brain Res. 275, 13-21. 38. Neugebauer U. P. and Schaible H.-G. (1988) Peripheral and spinal components of the sensitization of spinal neurons during an acute experimental arthritis. Agents and Actions 25, 234237.
c-fos after noxious stimulation of the nasal mucosa
641
39. Noback C. R. and Demarest R. J. (1981) The Human Nervous System Basic Principles ofNeurobiology. McGraw-Hill, Guatemala. 40. Noguchi K., Morita Y., Kiyama H., Sato M., Ono K. and Tohyama M. (1989) Preproenkephalin gene expression in the rat spinal cord after noxious stimuli. Molec. Brain Res. 5, 227-234. 41. Pellegrino L. J., Pellegrino A. S. and Cushman A. J.(1979) A Stereotaxic Atlas of the Rat Brain. Plenum Press, New York. 42. Qvist H., Deitrichs E., Rijste L. S. and Walberg F. (1983) A projection from the raphe nuclei to the lateral reticular nucleus in the cat. Archs ital. Biol. 121, 249-257. 43. Reeh P. W., Kocher L. and Jung S. (1986) Does neurogenic inflammation alter the sensitivity of unmyelinated nociceptors in the rat? Brain Res. 384, 42-50. 44. Ruda M. A., Cohen L., Shiosaka S., Takahashi O., Allen B., Humphrey E. and Iadarola M. J. (1989) In situ hybridization histochemical and immunocytochemical analysis of opioid gene products in a rat model of peripheral inflamation. In Processing of Sensory Information in the Superficial Dorsal Horn of the Spinal Cord (eds Cervero F., Bennett G. J. and Headley P. M.), pp. 365-382. Plenum Press, New York. 45. Sagar S. M., Sharp F. R. and Curran T. (1988) Expression of c-fos protein in brain: metabolic mapping at the cellular level. Science 240, 1328-1331. 46. Shigenaga Y., Nakatani Z., Nishimori T., Suemune S., Kuroda R. and Matano S. (1983) The cells of origin of cat trigeminothalamic projections: especially in the caudal medulla. Brain Res. 277, 201-222. 47. Sotgiu M. L. (1985) Inhibition of the nociceptive jaw opening reflex by lateral reticular nucleus (LRN) in the rabbit. Neurosci. L&t. (Suppl.) 22, S476. 48. Steen K. H., Anton F., Reeh P. W. and Handwerker H. 0. (1990) Senzitization and selection excitation by protons of nociceptive nerve endings in rat skin. Eur. J. Physiol. 415 (suppl. l), R106. 49. Weihe E., Iadarola M. J., Nohr D., Miiller S., Millan M. J., Yanaihara N., Stein C. and Herz A. (1991) Sustained expression and colocalization of proenkephalin and prodynorphin opioids and c-fos protein in dorsal horn neurons revealed in arthritic rats. Excerpta med. (in press). 50. Wiberg M., Westman J. and Blomqvist A. (1986) The projection to the mesencephalon from the sensory trigeminal nuclei. An anatomical study in the cat. Brain Res. 399, 5148. 51. Williams S., Pini A., Evan G. and Hunt S. P. (1989) Molecular events in the spinal cord following sensory stimulation. In Processing of Sensory Information in the Superficial Dorsal Horn of the Svinal Cord (eds Cervero F.. Bennett G. J. and Headley P. M.), pp. 273-284. Plenum Press; New York. ” _ 52. Willis W. D. (1985) The Pain Sysfem. Karger, Basel. 53. Woolf C. J. (1983) Evidence for a central component of postinjury pain hypersensitivity. Nafure 308, 686688. (Accepted 29 October 1990)
0306-4522/91
1991
$3.00 + 0.00
Pergamon Press plc 0 1991 IBRO
c-FOS-LIKE IMMUNOREACTIVITY IN RAT BRAINSTEM NEURONS FOLLOWING NOXIOUS CHEMICAL STIMULATION OF THE NASAL MUCOSA F. ANTON,*? T. HERDEGEN,~P. PEPPEL~ and J. D. LEAH$ tbrstitut fur Physiologie und Biokybernetik, Universitltsstr. 17, D-8520 Erlangen, F.R.G. $11. Physiologisches Institut, Im Neuenheimer Feld 326, D-6900 Heidelberg, F.R.G. Abstract-It has previously been shown that noxious and non-noxious peripheral stimuli induce c-fos expression in spinal dorsal horn neurons. In the present study we have examined the expression of c-fos in brainstem neurons following noxious chemical stimulation of the respiratory region of the nasal mucosa. In urethane-anaesthetized rats we injected mustard oil or applied CO, pulses to the right nasal cavity. In control animals we applied paraffin oil or a continous flow of air. A further group of control animals was anaesthetized and not subjected to any experimental treatment. Two hours after the first stimulus the rats were perfused with 4% phosphate-buffered paraformaldehyde. Brainstem sections were incubated with primary antiserum against the FOS protein and processed according to the ABC method. Only the mustard oil-treated rats had obvious signs of rhinitis and displayed FOS-positive cells in laminae I and II of the subnucleus caudalis and in the subnucleus interpolaris of the trigeminal brainstem nuclear complex as well as in the medullary lateral reticular nucleus. These areas are known to be involved in the processing of nociceptive information. Although CO, pulses applied to the nasal mucosa are known to evoke pain sensations in man we did not observe any FOS-positive neurons in trigeminal and reticular brainstem areas of CO,-treated rats. This lack of c-fos expression probably results from the fact that unlike mustard oil, CO, did not induce any apparent inflammatory reactions. In all animals c-fos expression was found in the nucleus of the solitary tract and in the area postrema. Staining in these areas might partly result from factors related to anaesthesia, changed respiration parameters and stress. Since the mustard oil-treated rats displayed the highest levels of immunoreactivity in the nucleus of the solitary tract and in the area postrema, additional effects specifically related to nociceptive input are very likely.
The ethmoidal nerve innervates the respiratory region of the nasal mucosa and is believed to constitute the afferent limb of protective upper airway reflexes. Within the trigeminal brainstem nuclear complex (TBNC) ethmoidal primary afferents have been shown in cat and rat to project exclusively to laminae I and II of subnucleus caudalis (SNC) and to portions of subnucleus interpolaris (SNI),‘,*’ areas which are known to contain numerous nociceptive neurons.9*‘5 Peripheral and central electrophysiological recordings in cat5*28,2g provide further support for the nociceptive function of the ethmoidal nerve. In rat, additional ethmoidal primary afferent terminals have been localized in the interstitial subnucleus of the nucleus tractus solitarius (NTS),* an area known to be involved in control of the respiratory rhythm.i4 Psychophysical investigations in humans have shown that noxious chemical stimulation of the nasal *To whom correspondence should be addressed. ABC, avidin-biotin complex; AP, area postrema; FOS-LI, FOS-like immunoreactivity; LRN, lateral reticular nucleus; NGS, normal goat serum; NTS, nucleus tractus solitarius; PB, phosphate buffer; PBS, phosphate-buffered saline; PBST, PBS with 0.2% Triton X- 100; SNC, subnucleus caudalis; SNI, subnucleus interpolaris; TBNC, trigeminal brainstem nuclear complex.
Abbreviations:
629
mucosa with capsaicin or CO, induces pain sensations and evokes protective reflexes like secretion and sneezing.‘2*22~23 The goal of the present study was to characterize this system further by mapping second and higher order brainstem neurons activated by noxious chemical stimulation of the nasal mucosa. A recently developed method to perform this mapping at the single cell level is based on the expression of the proto-oncogene c-fos which is triggered by extrinsic stimuli. The FOS protein is a nuclear protein participating in the control of the transcription rate of target genes which can play a role in long term alterations of cellular function.36 In spinal cord neurons FOS is induced by sensory stimulation.‘* More specifically, in superficial laminae of the dorsal horn, FOS rapidly appears following the application of noxious heat or chemical stimuli.‘8*25 Starting from the hypothesis that the ethmoidal nerve mainly carries nociceptive information we have examined the central projections of this nerve in the companion paper.* In the present study we used FOS-like immunoreactivity (FOS-LI) to map the distribution of brainstem neurons45 activated by noxious chemical stimulation of the nasal mucosa. These results have been partly described in a short communication.’
630
F. ANTON VI ul EXPERIMENTAL
PROCEDURES
Stimulating procedures
Twenty male Wistar rats weighing 320440 g were lightly anaesthetized with urethane (1 g/kg, i.p.) and divided into five experimental groups, according to the applied stimulation procedure. Mustard oil group (n = 6). In order to stimulate the ethmoidal nociceptors 10~1 of 5% mustard oil (allyl-isothiocyanate) in paraffin oil were injected into the nasal meatus. A blunt Hamilton syringe was used to apply liquid chemicals to the right nasal cavity (for further details see companion paper).? Control group 1 (n = 2). To control for non-specific effects related to the injection procedure, 10 pl of pure paraffin oil were injected into the nasal cavity. CO, group (n = 6). Noxious stimulation of the nasal mucosa was done with 100% CO,. To prevent long lasting hyperventilation we applied CO, pulses of 15 s duration followed by interstimulus intervals of 15 s rather than continuous flow of the stimulating gas. These pulses were presented in a humidity-saturated continuous airflow (20ml/s) adjusted to a temperature of 28°C. The whole stimulating procedure lasted 1 h. For gas application to the nasal mucosa a thin plastic tube was inserted into the right nasal cavity. Gas leaks were prevented by adjusting the tube’s diameter individually to seal the nostril. This tube was connected to an olfactometerz2.*r presenting a continuous airflow in which defined stimulating gas pulses can be applied without any changes in humidity, temperature and total flow rate. Control group 2 (n = 2). To control for non-specific effects related to the stimulating procedure described in the CO, group we presented a continuous similar airflow without any CO, application. Confrol group 3 (n = 4). To control for background FOS-LI and/or effects related to anaesthesia in certain brainstem areas, four animals were anaesthetized 2 h prior to the perfusion and did not undergo any further treatment. Tissue processing
Two hours after the initiation of the stimulation procedures the rats were deeply anaesthetized with thiobutabarbital sodium (Inactin”, 125 mg/kg, i.p.) and perfused through the aorta with 7&100 ml warm phosphate-buffered saline (PBS) containing 0.1% sodium nitrite and 10,000 units/l heparin followed by 50 ml of warm fixative (4% paraformaldehyde in PBS). The final cold flush consisted of 200 ml of the same fixative. The brains were removed and postfixed for &6 h in cold fixative containing 30% sucrose. They were then stored in a 30% sucrosephosphate buffer (PB: 0.1 M, pH 7.4) at 4°C for 3-8 days. Serial coronal brainstem sections (40 pm) were cut on a freezing microtome and processed for immunohistochemistry ai free floating sections. We used chilled uhosphate buffered saline with 0.2% Triton X- 100 (PBST)’ as diluent for all immunoreagents and rinsing solutions. The sections were first rinsed in PBST and then incubated in a blocking solution containing 1.5% normal goat serum (NGS) at 37°C for 1 h. The tissue was then incubated for 36 h in polyclonal rabbit antiserum directed against the c-fos protein (Medac, Hamburg), diluted 1: 1000. After that the tissue was processed using the avidin-biotin complex (ABC) method.” Following a final rinse the sections were developed in a solution of 0.01% diaminobenzidine and 0.02% hydrogen peroxide. The staining procedure was stopped by washing the tissue in doubly distilled water. The sections were then mounted on chrome-alum-gelatine coated slides, air dried and coverslipped. The brainstem sections were examined under a light microscope. The location of immunoreactive cell bodies was mapped with camera lucida drawings and microphoto-
graphs were taken. From each above, the rat with the highest chosen to screen the number of several distinct brainstem areas. were made by using a stereotaxic
of the groups described density of labelling WEiS c-fos-positive neurons in Anatomical correlations atlas.4’
RESULTS
The reactions of the animals to the stimulation procedures and the location of FOS-positive brainstem cells within the different groups exhibited the following features. Mustard
oil group
Immediately following mustard oil application all rats strongly expired through the nose and then stopped breathing for up to 15 s. Signs of inflammation like mucosal swelling, reddening and secretion could rapidly be observed and were still present when the animals were about to be perfused. As shown in camera lucida drawings of coronal sections through the brainstem (Fig. l), mustard oil treatment caused extensive FOS-LI in several brainstem nuclei. In the TBNC c-fos-positive cells could only be observed in the ipsilateral SNC and SNI. Labelling started about 0.7 mm caudal to the obex at the rostra1 pole of SNC and extended for a distance of about 1.4 mm in the rostra1 direction to the caudal pole of SNI. FOS-positive cell nuclei were strictly located in the superficial laminae (I and II) of the ventrolateral portion of SNC (see Figs 1 and 2a). In SNI the density of labelling decreased rapidly, stained nuclei tended to be situated more medially than in SNC and could also be observed in dorsal aspects of the subnucleus (Figs 1 and 2b). Counts across the ipsilateral SNC and SNI revealed up to 246 labelled cells (see Table 1). No signs of FOS-LI could be found in the subnucleus oralis or in the main sensory nucleus. c-fos-Positive could be observed neurons bilaterally in the NTS, as shown in Figs 1 and 3. Labelling started about 0.2 mm caudal to the obex and spanned a distance of about 1.8 mm in the rostra1 direction. Up to 402 stained cells were bilaterally counted across this nucleus, as shown in Table 1. A large number of stained cell nuclei (up to 225) could also be observed throughout the area postrema (AP) (see Figs I and 4). Finally, the mustard oil-treated rats displayed bilateral FOS-LI (up to 125 labelled cells, see Table 1) in the medullary lateral reticular nucleus (LRN), as shown in Figs 1 and 5. Control
group
Following apnoea or animals. We the trigeminal control rats.
1 the paraffin oil injection no signs of inflammation were observed in the could not detect any FOS-LI in any of or reticular brainstem nuclei of these Only NTS and AP displayed a rather
c-fos after noxious stimulation of the nasal mucosa
631
Fig. 1. Camera lucida drawings of coronal brainstem sections from a mustard oil-treated rat showing localization of FOS-positive neurons (dots) in different nuclei along the rostrocaudal axis. The dashed lines indicate subnuclei of the TBNC. (1) FOS-LI in SNC (a), in the transition zone between SNC and SNI (b) and in SNI (c). (2) FOS-positive cells in NTS, (3) in AP and (4) in LRN. PD, pyramidal decussation; ‘IT, trigeminal tract. Numbers to the right of each figure indicate the distance in millimetres rostra1 (+) or caudal (-) to the obex. low density of labelled cell nuclei (up to 48 and 72 cells respectively, see Table 1).
co,
group
The six rats stimulated with CO2 sneezed and decreased their respiration frequency following the presentation of the first CO2 stimuli. These reactions lasted 1-2 min and vanished during the course of the remaining stimulation procedure. No obvious signs of inflammation could be observed. In these animals the TBNC was devoid of FOS-LI. In addition no FOS-positive cells could be found in the reticular formation. In the caudal NTS bilateral labelling of cell nuclei (up to 195 cells) could be observed. The labelling was consistently less dense than in the mustard oil group. As displayed in Table 1, up to 130 FOS-positive cells were counted in AP, again considerably less than in the mustard oil group. Control group 2
In the two rats in which a continuous tIow of air was applied to the nasal mucosa we observed a slight twitching of the nose and face muscles which lasted some seconds following the start of the airflow presentation. In these control animals we did not find any FOS-positive neurons in TBNC and in the reticular formation. In NTS we observed up to 93 labeled nuclei, whereas up to 70 stained neurons were counted in AP.
Control group 3
The four rats which were anaesthetized and not subjected to any further treatment displayed a rather low density of FOS-LI in NTS (up to 65 cells, bilaterally) and in AP (up to 46 cells). No signs of labelling were observed in any of the other brainstem nuclei mentioned above (see Table 1).
DISCUSSION
FOS-like immunoreactivity in trigeminal brainstem nuclear complex and lateral reticular nucleus
In this study two different noxious chemical stimuli, mustard oil and CO, were applied to the nasal mucosa of anaesthetized rats. In rat, mustard oil has been shown to excite polymodal C-nociceptors and to cause neurogenic inflammation which in turn leads to sustained firing for up to 30 min and to enhanced thermal responsiveness of these units.43 In humans, topical application of mustard oil to the skin leads to a burning pain sensation and to local inflammatory responses which persist for several minutes following washout of the application site.” CO2 pulses applied to the nasal mucosa have been reported to induce pain in man.22*23In a rat skinsaphenous nerve in vitro preparation, electrophysiological recordings revealed that CO2 specifically
F. ANTON et al.
Fig. 2a.
634
F. Awrorj
excites nociceptive primary afferents and that this effect can probably be attributed to lowering of the intracellular pH to acid levels.48 In these experiments the nociceptors displayed an ongoing activity as long as CO, was applied to the receptive fields, Human
et ai.
psychophysical experiments in our laboratory revealed, however, that long lasting CO, pulses (up to 20 s) applied to the nasal mucosa induce very phasic pain sensations never lasting more than 34 s (unpubIished observations). Under these conditions the
Fig.
?a
c-fos after noxious stimulation of the nasal mucosa
Fig. 3b. Fig. 3. Photographs of coronal sections displaying immunoreactive cell nuclei in caudal (a) and rostra1 (b) NTS of a mustard oil-treated rat. x 119. Boxes in the schematic drawings indicate areas shown in the photomicrographs.
635
635
Fig. 4. Fh~t~mi~r~~a~h displa$ng RX-positive cells in AP following mustard oif treatment. x 119. The area shown in the ptrotograph is indicated by the box in the schematic drawing.
secretion of mucus induced by noxious stimulation of the nasal mucosa probably results in fast buffering of CO, related pW changes.
In the present study the animals treated with mustard oil displayed c-fos-positive cells in the ~rigern~n~l SNC and SNI as well as in the lateral
c-fos after noxious stimulation of the nasal mucosa
Fig. 5. Photograph of a coronal section showing FOS-LI in LRN of a mustard oil-treated rat. x 119. The box in the schematic drawing indicates the photographed area.
637
F. ANTON et al.
638 Table
1. Numbers
of immunoreactive brainstem nuclei Brainstem
Experimental group Mustard Oil Control 1 co, Control 2 Control 3
cells
in different
nuclei
TBNC
LRN
NTS
AP
246
12s
402 48 195 93 65
22s 72 130 70 46
-
Counts from the rat displaying labelling in each experimental
the highest density group are shown.
of
reticular nucleus (LRN), whereas in the CO,-treated rats these nuclei were devoid of labelling. This discrepancy can probably be explained by the differences of the physiological reactions evoked by mustard oil and CO* application. The mustard oil-treated rats showed obvious signs of inflammation, like mucosal swelling, reddening and secretion. As discussed above, this neurogenic inflammation probably caused an ongoing activity in ethmoidal nociceptive primary afferents and resulted in a high degree of synaptic input in higher order neurons. In contrast, the CO, pulses used in our study probably induced rather phasic discharges of the ethmoidal nociceptive primary afferents. In addition no signs of inflammation could be observed in these animals. It is thus very likely that the summation of synaptic input to the respective higher order neurons was low compared to the mustard oil condition. Since the amount of FOS protein produced is probably directly related to the degree of synaptic activation5’ we conclude that the lack of FOSpositive neurons in the TBNC and LRN of CO,treated animals is likely to be due to comparatively low activity levels of central neurons under these conditions. Although the function of the FOS protein is not yet known, it is believed to participate in long lasting functional alterations of neurons.‘8s25 Such long term changes of neuronal function could be particularly relevant in tonic, inflammatory types of pain which can, for example, trigger persistent central mechanisms of sensitization.‘633853 Since none of the control groups displayed any FOS-LI in TBNC or LRN we can conclude (1) that nasal mucosa stimuli which do not evoke noxious input or only lead to brief, phasic discharges of nociceptive primary afferents do not induce c-fos expression in the trigeminal system or in the lateral reticular formation and (2) that no background labelling related to our anaesthetic conditions can be observed in these brainstem areas (concerning depressing effects of anaesthesia see Basbaum et ~f.).~ Within TBNC the localization of c-fos-positive cells corresponded exactly to the location of the ethmoidal primary afferent terminals.’ Thus it is very likely that the majority of stained cells were driven by monosynaptic ethmoidal input. The lack of a significant population of polysynaptically excited cells in
areas not receiving primary afferent input could be related to anaesthesia.4 However, since our rats were lightly anaesthetized and still showed pronounced nociceptive reflexes we probably would not have missed a whole population of polysynaptically activated cells. One major function of the TBNC areas containing FOS-LI is the processing of nociceptive information (see companion paper).2 Spinal dorsal horn and SNC lamina I neurons project to many brainstem and diencephalic nuclei including dorsal and ventral areas of the reticular formation,35a46 nucleus tractus solitarius,32 parabrachial area,‘9~s0hypothalamus’ and to medial and ventrolateral thalamic nuclei (for review see Willis).‘* Projections of nociceptive SNI neurons have not been examined as extensively. To gain further insight into functional aspects it would be interesting to know whether all of these areas, or preferentially some, receive input from highly activated trigeminal nociceptive neurons. Menetrey et ~1.~’performed a double labelling study at the level of the spinal dorsal horn and found a significant portion of FOS-positive neurons projecting to the areas mentioned above. Differential injections of retrograde tracer into single nuclei of the brain will increase our knowledge about projections and functions of FOS-positive nociceptive brainstem neurons. Recent reports indicate that inflammatory reactions which lead to sensitized responses of nociceptive primary afferents and hence to significant peptide release from their central terminalsi might be required to induce c-fos expression in the superficial spinal dorsal horn. Intrathecal application or superfusion of the spinal cord with neuropeptides like substance P, neurokinin A or bombesin caused c-fos staining in superficial laminae of the dorsal horn.25a5’ Williams et al.” reported that, independent of the nature of the cutaneous noxious stimulation, similar levels of c-fos immunoreactivity in the spinal dorsal horn are obtained if the stimuli lead to comparable intensities of inflammation as expressed by plasma extravasation and oedema. Furthermore, the pattern of FOS-LI in the superficial dorsal horn is coincident with the distribution of neurons that exhibit increased preproenkephalin or preprodynorphin mRNA levels in rat models of Moreover in arthritic peripheral inflammation. 20,40,44 rats, Weihe and co-workers49 have shown extensive co-localization of proenkephalin or prodynorphin opioids and FOS protein in the dorsal horn. The fact that FOS could regulate the preproenkephalin and preprodynorphin genes under these conditions (see review paper by Morgan and Curran) stresses the importance of this nuclear protein in long term CNS processing of nociceptive input from inflamed tissue. In the present study the mustard oil-treated rats were the only ones displaying FOS-LI in the medullary LRN. This nucleus has been reported to receive input from spinal second order neurons via
c-fos
after noxious stimulation of the nasal mucosa
the lateral spinoreticular tract.35 The induction of in this nucleus thus resulted from polysynaptic activation of the respective neurons, which is a further indication that the urethane anaesthesia used in our study did not generally prevent c-fos expression in cells with polysynaptic input. LRN is known to be involved in sensorimotor integration because it is highly interconnected with structures like motor cortex, cerebellum, red nucleus, ventral and dorsal horns of the spinal cord and other structures.6~24~26~3’~34~35~42 The labelling observed in our study could thus be related to persistent modulation of motor reactions like facilitation of protective reflexes triggered by tonic noxious input. Concerning processing of nociceptive information, the LRN has more recently been reported to be involved in descending inhibition.“*‘3,37s47In rat, Janss and Gebhart*’ have proposed that spinal alpha, and serotonin receptors mediate the inhibition of nociceptive responses from the LRN. Alternatively to the modulation of motor reactions discussed above, the c-fos-positive LRN neurons observed in our study could thus reflect sustained descending inhibition of nociceptive input. At the moment we do not have any explanation for the fact that none of the nuclei in the rostra1 ventral medulla (see Basbaum and Fields),3 involved in the descending control of spinal nociceptive processing, displayed FOS-LI.
c-fos
C-fos like immunoreactivity in nucleus tractus solitarius and area postrema
The NTS is roughly divided into a medial and a lateral region. The medial part mainly receives visceral afferent input and is involved in integration of respiratory and cardiovascular parameters.’ The lateral part predominantly receives gustatory input.r4 The AP constitutes the caudal ventral floor of the fourth ventricle and contains chemosensitive neurons involved in respiratory contro13’ The animals of all five groups described above displayed at least a moderate amount of FOS-LI in these areas. The lowest numbers of labelled cells were found in the control groups. This finding could reflect a low background expression of the c-fos gene. Other authors have described scattered FOS-positive brainstem neurons in non-stimulated animals.45 Since the FOS-positive cells in our study were restricted to
639
NTS and AP it is more likely that effects related to anaesthesia, like changes in pulse rate, respiration frequency and volume, were involved. The highest densities of FOS-LI in NTS and AP were observed in the rats treated with noxious chemical stimuli, and the mustard oil-treated animals displayed a higher density than the CO,-stimulated ones. In both groups we observed protective upper airway reflexes like apnoea, sneezing and gasping. The dramatic changes in respiratory and cardiovascular parameters resulting from the noxious stimultion could have induced a more pronounced c-fos expression than in the control groups. The inhalation of CO2 can lead to an acidosis which in turn causes hyperventilation. To limit c-fos expression induced by these respiratory changes we did not apply the CO2 continuously but in the form of pulses. Consequently we did not see any signs of sustained hyperventilation during the CO2 stimulation. Since the mustard oil-treated rats were the only ones reacting with apnoea for a longer period, this factor could explain the highest density of labelling found in these animals. Concerning NTS, it is possible that the tonic noxious stimulation of ethmoidal primary afferents in the mustard oil group increased the amount of c-fos expression. In the interstitial subnucleus of NTS, c-fos expression could also have resulted from monosynaptic activation. This subnucleus, which is specifically involved in respiratory integration, has been shown to contain terminals of ethmoidal primary afferents (see Anton and Peppel).’ Menetrey and Basbaum3* showed presumably nociceptive spinal and medullary dorsal horn neurons to project to NTS and thus probably to be involved in somatovisceral and viscerovisceral reflex activation. Therefore the expression of c-fos is likely to have been induced by polysynaptic activation in a large population of NTS neurons. Finally, we cannot exclude that mustard oil as well as CO, could have reached branches of the cranial nerves IX and X via the pharynx, larynx and oral cavity (see Ref. 2). Since both substances cause taste sensations, gustatory induction of c-fos might have been possible in a population of NTS neurons. Acknowledgements-We would like to thank Mr K. Burian for excellent technical assistance. This work was supported by the DFG grant An 158/1-l.
REFERENCES
Anton F., Herdegen T., Peppel P. and Leah J. D. (1989) C-fos expression in rat brainstem neurones following noxious stimulation of the nasal mucosa. Eur. J. Neurosci. (Suppl.) 2, 184. Anton F. and Peppel P. (1991) Central projections of trigeminal primary a&rents innervating the nasal mucosa: a horseradish peroxidase study in the rat. Neuroscience 41, 617628. Basbaum A. I. and Fields H. L. (1984) Endogenous pain control systems: brainstem spinal pathways and endorphin circuitry. A. Rev. Neurosci. 7, 309-338. Basbaum A. I., Presley R., Menttrey D., Chi S. J. and Levine J. D. (1989) Somatic, articular and visceral noxious-stimulus-evoked expression of c-fos in the spinal cord of the rat: differential patterns of activity and modulation by analgesic agents. In Processing of Sensory Information in the Superficial Dorsal Horn of the Spinal Cord (eds Cervero F., Bennett G. J. and Headley P. M.), pp. 3655382. Plenum Press, New York.
640
F. ANTONet al.
5. Beuerman R. W. (1975) Neurons in trigeminal nucleus and reticular formation excited by ethmoidal nerve stimulation. Brain Res. 92, 479484. 6. Brodal P., Marsala J. and Brodal A. (1967) The cerebral cortical projection to the lateral reticular nucleus in the cat, with special reference to the sensorimotor cortical areas. Brain Res. 6, 252-274. 7. Burstein R., Cliffer K. D. and Giesler G. J., Jr (1987) Direct somatosensory projection from the spinal cord to the hypothalamus and telencephalon. J. Neurosci. 7, 4159-4164. 8. Bystrzycka E. K. and Nail B. S. (1985) Brainstem nuclei associated with respiratory, cardiovascular and other autonomic functions. In The Rat Neroous System (ed. Paxinos G.), Vol. 2, pp. 95-110. Academic Press, Sydney. 9. Dubner R. and Bennett G. J. (1983) Spinal and trigeminal mechanisms of nociception. A. Reu. Neurosci. 6, 381418.
10. Duggan A. W., Morton C. R., Hendry A. I. and Hut&son W. D. (1989) Peripheral stimuli releasing neuropeptides in the dorsal horn of the cat. In Processing of Sensory Information in the Superficial Dorsal Horn of the Spinal Cord (eds Cervero F., Bennett G. J. and Headley P. M.), pp. 347-364. Plenum Press, New York. 11. Gebhart G. F. and Ossinov M. (1986) Characterization of inhibition of the sninal nociceutive tail-flick reflex in the rat from the medullary lateral reticular nucleus. J. Neurosci. 6, 701-713. _ 12. Geppetti P., Fusco B. M., Marabini S., Maggi C. A., Fan&ha& M. and Sicuteri F. (1988) Secretion, pain and sneezing induced by the application of capsaicin to the nasal mucosa in man. Br. J. Pharmac. 93, 509-514. 13. Hall J. G., Duggan A. W., Morton C. R. and Johnson S. M. (1982) The localization of brainstem neurons tonically inhibiting dorsal horn neurons of the cat. Brain Res. 244, 215-222. 14. Hamilton R. B. and Norgren R. (1984) Sensory projections of gustatory nerves in the rat. J. camp. Neurol. 222, X%577. 15. Havashi H.. Sumino R. and Sessle B. J. (1984) Functional organization of trigeminal subnucleus interpolaris: no&eptive ‘and innocous afferent inputs, projections to thalamis, cerebellum, and spinal cord, and descending modulation from periaqueductal gray. J. Neurophysiol 51, 890-908. 16. Hoheisel U. and Mense S. (1989) Long-term changes in discharge behaviour of cat dorsal horn neurones following noxious stimulation of deep tissues. Pain 36, 239-249. 17. Hsu S. M., Raine L. and Fanger H. (1981) Use of avidin-biotin-peroxidase complex (ABC) in immunoperoxidase techniques. J. Histochem. Cyrochem. 29, 577-580. 18. Hunt S. P., Pini A. and Evan G. (1987) Induction of c-fos-like protein in spinal cord neurons following sensory stimulation. Nature 328, 632634. 19. Hylden J. L. K., Anton F. and Nahin R. L. (1989) Spinal lamina I projection neurons in the rat: collateral innervation of parabrachial area and thalamus. Neuroscience 28, 27-37. 20. Iadarola M. J., Douglass J., Civelli 0. and Naranjo J. R. (1988) Differential activation of spinal cord dynorphin and enkephalin neurons during hyperalgesia: evidence using cDNA hybridisation. Brain Res. 455, 205-212. 21. Janss A. J. and Gebhart G. F. (1987) Spinal monoaminergic receptors mediate the antinociception produced by glutamate in the medullary lateral reticular nucleus. J. Neurkci. 7, 2862-2873. 22. Kobal G. (1985) Pain-related electrical potentials of the human nasal mucosa elicited by chemical stimulation. Pain 22, 151-163. 23. Kobal G. and Hummel C. (1988) Cerebral chemosensory evoked potentials elicited by chemical stimulation of the olfactory and respiratory nasal mucosa. Electroenceph. clin. Neurophysiol. 71, 241-250. 24. Kiinzle H. and Wiesendanger M. (1974) Pyramidal connections to the lateral reticular nucleus in the cat: a degeneration study. Acta anat. 88, 105-114. 25. Leah J. D., Herdegen T. and Zimmermann M. (1989) Physiological and pharmacological induction of c-fos protein immunoreactivity in superficial dorsal horn neurons. In Processing of Sensory Information in the Superficial Dorsal Horn of the Spinal Cord (eds Cervero F., Bennett J. G. and Headley P. M.), pp. 307-310. Plenum Press, New York. 26. Lowey A. D., Wallach J. H. and McKellar S. (1981) Efferent connections of the ventral medulla oblongata in the rat. Brain Res. Rev. 3, 63-80.
27. Lucier G. E. and Egizii R. (1986) Central projections of the ethmoidal nerve of the cat as determined by the horseradish peroxidase tracer technique. J. camp. Neural. 247, 123-132. 28. Lucier G. E. and Egizii R. (1988) Characterization and projection of nociceptive input from the nasal cavity of the cat. In Proceedings of the Vth World Congress on Pain (eds Dubner R., Gebhart G. F. and Bond M. R.), pp. 541-547. Elsevier, Amsterdam. 29. Lucier G. E. and Egizii R. (1989) Characterization of cat nasal afferents and brainstem neurones receiving ethmoidal input. Expl Neurol. 103, 83-89. 30. Magerl W., GrImer G. and Handwerker H. 0. (1990) Sensations and local inflammatory responses induced by application of carbachol, dopamine, 5-HT, histamine and mustard oil to the skin in humans. Eur. J. Physiol. 415 (suppl. I), R107. 31 Marchand J. E. and Hagino N. (1983) Afferents to the periaqueductal gray in the rat: a horseradish peroxidase study. Neuroscience 9, 95-106.
32. Menetrey D. and Basbaum A. I. (1987) Spinal and trigeminal projections to the nucleus of the solitary tract: a possible substrate for somatovisceral and viscerovisceral reflex activation. J. camp. Neurol. 25S, 439450. 33. Menetrey D., Gannon A., Levine J. D. and Basbaum A. I. (1989) The expression of c-fos protein in presumed nociceptive interneurons and projection neurons of the rat spinal cord: anatomical mapping of the central effects of noxious somatic, articular and visceral stimulation. J. camp. Neurol. 285, 177-195. 34. Menetrey D., De Pommery J. and Besson J. M. (1984) Electrophysiological characteristics of lumbar spinal cord neurons backfired from lateral reticular nucleus in cat. J. Neurophysiol. 52, 595611. 35. Menetrey D., Roudier F. and Besson J. M. (1983) Spinal neurons reaching the lateral reticular nucleus, as studied in the rat by retrograde transport of horseradish peroxidase. J. camp. Neurol. 220, 439452. 36. Morgan J. E. and Curran T. (1989) Stimulus-transcription coupling in neurons: role of cellular immediate-early genes. Tren& Neurosci. 12(1l), 459462. 37. Morton C. R., Johnson S. M. and Duggan A. W. (1983) Lateral reticular regions and the descending control of dorsal horn neurons of the cat: selective inhibition by electrical stimulation. Brain Res. 275, 13-21. 38. Neugebauer U. P. and Schaible H.-G. (1988) Peripheral and spinal components of the sensitization of spinal neurons during an acute experimental arthritis. Agents and Actions 25, 234237.
c-fos after noxious stimulation of the nasal mucosa
641
39. Noback C. R. and Demarest R. J. (1981) The Human Nervous System Basic Principles ofNeurobiology. McGraw-Hill, Guatemala. 40. Noguchi K., Morita Y., Kiyama H., Sato M., Ono K. and Tohyama M. (1989) Preproenkephalin gene expression in the rat spinal cord after noxious stimuli. Molec. Brain Res. 5, 227-234. 41. Pellegrino L. J., Pellegrino A. S. and Cushman A. J.(1979) A Stereotaxic Atlas of the Rat Brain. Plenum Press, New York. 42. Qvist H., Deitrichs E., Rijste L. S. and Walberg F. (1983) A projection from the raphe nuclei to the lateral reticular nucleus in the cat. Archs ital. Biol. 121, 249-257. 43. Reeh P. W., Kocher L. and Jung S. (1986) Does neurogenic inflammation alter the sensitivity of unmyelinated nociceptors in the rat? Brain Res. 384, 42-50. 44. Ruda M. A., Cohen L., Shiosaka S., Takahashi O., Allen B., Humphrey E. and Iadarola M. J. (1989) In situ hybridization histochemical and immunocytochemical analysis of opioid gene products in a rat model of peripheral inflamation. In Processing of Sensory Information in the Superficial Dorsal Horn of the Spinal Cord (eds Cervero F., Bennett G. J. and Headley P. M.), pp. 365-382. Plenum Press, New York. 45. Sagar S. M., Sharp F. R. and Curran T. (1988) Expression of c-fos protein in brain: metabolic mapping at the cellular level. Science 240, 1328-1331. 46. Shigenaga Y., Nakatani Z., Nishimori T., Suemune S., Kuroda R. and Matano S. (1983) The cells of origin of cat trigeminothalamic projections: especially in the caudal medulla. Brain Res. 277, 201-222. 47. Sotgiu M. L. (1985) Inhibition of the nociceptive jaw opening reflex by lateral reticular nucleus (LRN) in the rabbit. Neurosci. L&t. (Suppl.) 22, S476. 48. Steen K. H., Anton F., Reeh P. W. and Handwerker H. 0. (1990) Senzitization and selection excitation by protons of nociceptive nerve endings in rat skin. Eur. J. Physiol. 415 (suppl. l), R106. 49. Weihe E., Iadarola M. J., Nohr D., Miiller S., Millan M. J., Yanaihara N., Stein C. and Herz A. (1991) Sustained expression and colocalization of proenkephalin and prodynorphin opioids and c-fos protein in dorsal horn neurons revealed in arthritic rats. Excerpta med. (in press). 50. Wiberg M., Westman J. and Blomqvist A. (1986) The projection to the mesencephalon from the sensory trigeminal nuclei. An anatomical study in the cat. Brain Res. 399, 5148. 51. Williams S., Pini A., Evan G. and Hunt S. P. (1989) Molecular events in the spinal cord following sensory stimulation. In Processing of Sensory Information in the Superficial Dorsal Horn of the Svinal Cord (eds Cervero F.. Bennett G. J. and Headley P. M.), pp. 273-284. Plenum Press; New York. ” _ 52. Willis W. D. (1985) The Pain Sysfem. Karger, Basel. 53. Woolf C. J. (1983) Evidence for a central component of postinjury pain hypersensitivity. Nafure 308, 686688. (Accepted 29 October 1990)