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NMDA receptor subunit NR1-immunoreactivity in the rat pons and brainstem and colocalization with Fos induced by nasal stimulation
Brain Research 809 Ž1998. 221–230
Research report
NMDA receptor subunit NR1-immunoreactivity in the rat pons and brainstem and colocalization with Fos induced by nasal stimulation Mathias Dutschmann, Axel Guthmann, Horst Herbert
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Department of Animal Physiology, UniÕersity of Tubingen, Auf der Morgenstelle 28, D-72076 Tubingen, Germany ¨ ¨ Accepted 25 August 1998
Abstract In the present study, we examined the distribution of neurons in the parabrachial nucleus ŽPB., the Kolliker–Fuse nucleus ŽKF., the ¨ spinal trigeminal nucleus caudalis ŽSp5C., the nucleus of the solitary tract ŽNTS. and the ventrolateral medulla ŽVLM., which are activated by evoking the nasotrigeminal reflex and which exhibit immunoreactivity for the N-methyl-D-aspartate ŽNMDA. receptor subunit NR1. By stimulating the nasal mucosa with saline, we induced the expression of the immediate early gene c-fos and combined the immunocytochemical detection of the Fos protein with the detection of the NR1 subunit. Cell counts revealed that nasal stimulation, compared to anesthesia controls, resulted in highly significant increases Ž p F 0.001. of Fos-immunoreactive Ž-ir. neurons in the midlevel KF, the external lateral PB, and the Sp5C. In the central lateral PB, the rostral ventrolateral medulla including the Botzingerrpre-Botzinger ¨ ¨ complex, and in the ventrolateral and commissural NTS the increases were only moderately significant Ž p F 0.05.. With respect to the numbers of NR1-rFos-ir double-labeled neurons, significant increases were only observed in a subset of these pontomedullary nuclei. Increases were highly significant in the Sp5C Ž p F 0.001. and the midlevel KF Ž p F 0.01. and moderately significant Ž p F 0.05. in the external lateral PB, Botzingerrpre-Botzinger complex, and ventrolateral NTS. The present study revealed that nasotrigeminally activated ¨ ¨ neurons in mandatory and potential relay sites of the nasotrigeminal reflex circuit express the NR1 subunit. This finding strongly suggests that NMDA-type glutamate receptors are involved in the mediation of the nasotrigeminally evoked cardiovascular and respiratory responses. q 1998 Elsevier Science B.V. All rights reserved. Keywords: Parabrachial; Kolliker–Fuse; Solitarius; Trigeminal; Diving response; Sensory–autonomic integration ¨
1. Introduction Noxious stimulation of the nasal mucosa elicits a vital defensive reflex characterized by sudden cessation of breathing, bradycardia and peripheral vasoconstriction leading to a rise in arterial blood pressure w22x. These autonomic responses have been reported for several species w1,22,23,29x and are prominently expressed in diving mammals w13x. Sensory information from the nasal mucosa reaches the CNS via the ethmoidal nerve ŽEN5., a branch of the ophthalmic division of the trigeminal nerve w36,37x. Anatomical and pharmacological studies demonstrated that the first central relay site for EN5 afferents lies within the pars caudalis of the spinal trigeminal nucleus ŽSp5C. w2,24,25,30,32x. Furthermore, it was shown that the pontine ŽKF. nucleus respiratory neurons of the Kolliker–Fuse ¨ ) Corresponding author. Fax: q49-7071-292618; E-mail: [email protected]
w6,8x, which receive extensive projections from the Sp5C w14,31x, are also mandatory relays for the respiratory responses evoked by EN5 stimulation w9x. Most recently, we demonstrated that injections into the KF of the NMDA receptor antagonist AP5 blocked the EN5-evoked cardiorespiratory responses, indicating a major role for NMDA receptors in the mediation of the nasotrigeminal reflex w11x. In the present study, we investigated the potential involvement of NMDA receptors in other brain areas for mediating the nasotrigeminal reflex responses. By means of the stimulation triggered expression of the immediate early gene c-fos, we first identified neurons in pons and medulla that were activated after evoking the nasotrigeminal reflex w10x. We then stained the sections for the NMDA receptor-NR1 subunit to see if nasotrigeminally driven neurons also express NMDA receptors. Our findings may provide the basis for pharmacological experiments to further characterize the functional role of NMDA receptors
0006-8993r98r$ - see front matter q 1998 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 6 - 8 9 9 3 Ž 9 8 . 0 0 8 8 5 - 3
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Table 1 Summary of cell counts Žmean " S.E.M.. of Fos-ir neurons and NR1-rFos-ir double-labeled neurons in selected nuclei of the PBrKF complex after nasal stimulation with saline Ž n s 5. and after anesthesia control Ž n s 3. KF and PB nuclei
KF rostral aspects KF midlevel KF caudal aspects Superior lateral PB Central lateral PB External lateral PB
Fos-ir neurons
NR1-rFos-ir double-labeled neurons % Fos-ir neurons expressing NR1-ir
Stimulated
Control
Stimulated
Control
ŽStimulated group.
38 " 4) 239 " 16))) 33 " 7 49 " 5 104 " 16) 177 " 9)))
23 " 3 43 " 3 10 " 3 36 " 3 34 " 1 40 " 2
18 " 2 152 " 9)) 13 " 3 28 " 4 28 " 6 54 " 8)
16 " 2 23 " 5 8"3 23 " 3 20 " 1 25 " 1
47% 64% 34% 57% 27% 31%
for mediating the nasotrigeminal reflex response in different brain areas.
2. Materials and methods The investigations were carried out in accordance with the ethical guidelines for the care and use of experimental animals and were approved by the local council of animal care. We used male Wistar rats weighing 300–350 g which were housed under identical conditions in a room with a regular light–dark-cycle Ž06:00 h lights on, 18:00 h lights off. and food and water ad libitum. Rats used for the experiments were quickly removed from their home cages, anesthetized and then transferred to the laboratory. The data presented were derived from a total of eight rats in which the experimental manipulations and the immunocytochemical detection of the Fos protein and the NMDA receptor-NR1 subunit were successfully performed. Rats in which any step of the experimental protocol differed from the regular procedure were excluded from further analysis. 2.1. Experimental procedures To investigate the distribution of Fos- and NR1-rFosimmunoreactive Ž-ir. neurons in the pons and brainstem, we analyzed the brains from two experimental groups. In the first group, five rats were anesthetized with a mixture
of a-chloralose Ž150 mg kgy1 . and urethane Ž60 mg kgy1 . injected i.p. and placed on a heating pad Ž368C. to maintain body temperature. A small polyethylene tubing was inserted into the right nasal cavity and 0.2–0.3 ml saline at room temperature were infused. The saline infusion generally causes a pronounced suppression of breathing w10x. After 10 s, the saline was removed from the nasal cavity by suction. This stimulus was presented every 5 min for 1 h. The animals were killed 1.5 h after the last stimulation was given. The second group of rats Ž n s 3. served as controls. These rats were anesthetized as described above and then kept for 2.5 h on the heading pad in the surgery room. Thereafter, they were killed by transcardial perfusion and their brains were removed. For further details and extensive control experiments, see also Ref. w10x. 2.2. Tissue preparation At the end of the experiments, the stimulated rats were injected with a lethal dose of urethane Ž60 mg kgy1 . and transcardially perfused with 30 ml of phosphate buffered saline ŽPBS, 0.01 M at pH 7.4., followed by 500 ml of fixative containing 4% paraformaldehyde, 7.85 g lysine and 1.075 g sodium-meta-periodate in PBS. The brains were removed from the skull, postfixed in the same fixative for 1 h, placed in PBS containing 30% sucrose and stored at 48C. Coronal sections were cut at 40 mm on a freezing microtome and divided into three series. One
Table 2 Summary of cell counts Žmean " S.E.M.. of Fos-ir neurons and NR1-rFos-ir double-labeled neurons in selected nuclei of the medullary brainstem after nasal stimulation with saline Ž n s 5. and after anesthesia control Ž n s 3. Medullary nuclei
Sp5C A5-region Botzingerrpre-Botzinger ¨ ¨ RVLM CVLM Ventrolateral NTS Dorsomedial NTS Medial NTS Commissural NTS
Fos-ir neurons
NR1-rFos-ir double-labeled neurons % Fos-ir neurons expressing NR1-ir
Stimulated
Control
Stimulated
Control
ŽStimulated group.
261 " 40))) 24 " 3 40 " 7) 52 " 6) 41 " 5 109 " 21) 72 " 16 99 " 18 80 " 9)
5"1 27 " 6 12 " 2 24 " 3 31 " 12 30 " 19 68 " 14 104 " 21 46 " 5
139 " 18))) 13 " 2 32 " 8) 34 " 6 21 " 3 51 " 8) 16 " 3 41 " 9 33 " 4
3"1 18 " 6 8"2 20 " 4 25 " 12 17 " 11 17 " 4 43 " 14 25 " 1
53% 54% 80% 66% 52% 47% 21% 41% 41%
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series of sections was stained for Nissl-substance with thionine, the other two series were processed immunocytochemically. For immunocytochemistry, the sections were rinsed in Tris-buffered saline ŽTBS, 0.05 M at pH 7.6.. Nonspecific binding sites were blocked by incubating the sections in a solution containing 10% normal swine serum and 0.3% Triton X-100 in TBS. Thereafter, the sections were incubated in a sheep anti-Fos antiserum ŽCambridge Research Biochemicals, Northwich, UK. diluted 1:1000 in a carrier solution containing 2% normal swine serum and 0.3% Triton X-100 in TBS. After 48 h at 48C, the sections were rinsed in TBS and incubated for 1.5 h at room temperature in a biotinylated donkey anti-goat IgG antiserum ŽJackson ImmunoResearch, Dianova, Hamburg, Germany. diluted 1:1000 in carrier. Then, the sections were washed again in TBS and incubated for 1.5 h at room temperature in Avidin-Neutralite HRP ŽMolecular Probes Europe, Leiden, The Netherlands. diluted 1:1000 in carrier. After several rinses in TBS, the sections were incubated in 0.02% 3,3X-diaminobenzidine tetrahydrochloride ŽDAB; Sigma-Aldrich, Deisenhofen, Germany. in the presence of 0.01% H 2 O 2 and 0.3% Ni-ammonium sulfate ŽpH 8. for 10–30 min at room temperature, resulting in a black reaction product. The reaction was terminated by transferring the sections into TBS. For detection of the second antigen, the sections were washed again in TBS and incubated for 1 h in TBS containing 10% normal swine serum. Triton X-100 was omitted in all subsequent steps of the protocol. The sections were then incubated in a mouse anti-NMDA-NR1 antibody ŽPharMingen Deutschland, Hamburg, Germany.. This antibody is directed against a highly conserved amino acid sequence Ž660–811. and, thus, recognizes all isoforms of the NR1 subunit w38x. The sections were incubated in the primary antibody, diluted 1:200 in carrier containing 2% swine serum. After 48 h at 48C, the sections were rinsed in TBS and placed for 1 h at room temperature in a biotinylated goat anti-mouse IgG antiserum ŽJackson. diluted 1:200 in carrier solution, followed by repeated washes and incubation in a peroxidase-labeled avidin–biotin complex ŽABC, Vectastain Elite kit, Vector Laboratories, Burlingame, CA, USA.. Following rinses in TBS, the sections were incubated in 0.05% DAB in the presence of 0.01% H 2 O 2 for 10–30 min at room temperature, resulting in a brown reaction product. The reaction was terminated by transferring the sections in TBS. The sections were then mounted on gelatin-coated slides, air-dried, dehydrated in alcohols, cleared in xylene, and coverslipped with Entellan ŽMerck, Darmstadt, Germany.. Fig. 1. Camera lucida drawings of coronal sections through the dorsal pons ŽA–D, from rostral to caudal. illustrating the distribution of Fos-ir Žfilled circles. and NR1-rFos-ir double-labeled Žopen squares. neurons in the nuclei of the PBrKF complex. Sections shown in A–D correspond approximately to plates 53–56 of Paxinos and Watson w34x. Scale bar s 500 mm.
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2.3. Data analysis
3. Results
The sections were analyzed with a light microscope ŽReichert-Jung, Leica, Bensheim, Germany.. The distribution of Fos-ir neurons and neurons immunoreactive for Fos and NR1 protein ŽNR1-rFos-ir. was plotted with a camera lucida in the PBrKF nuclei and in the medullary brainstem. Cytoarchitectural boundaries of PBrKF and medullary nuclei were defined by superimposing the adjacent thionine-stained sections with the camera lucida drawings. The criteria for a neuron to be considered as doublelabeled were such that they had to exhibit a clearly blackstained cell nucleus and, in addition, a brown-stained cell body with clearly recognizable brown-stained primary dendrites. Representative examples of single and doublelabeled neurons were photographed. To evaluate the numbers of Fos-ir neurons and NR1-rFos-ir double-labeled neurons, we performed cell counts ipsilaterally in the brains of five stimulated rats and three control rats Žsummarized in Tables 1 and 2.. In brief, we counted cells in every third section through the pons and medulla. For the PBrKF, a total of eight sections through different rostrocaudal levels were analyzed: two sections from rostral levels Žcorresponding to the level shown in Fig. 1A., four sections from the midlevel ŽFig. 1B,C. and two sections from caudal levels ŽFig. 1D.. Separate cell counts were performed for the KF and the superior lateral, central lateral and external lateral PB. For the Sp5C, we analyzed three sections through the Sp5C at the level of the area postrema and just caudal to it ŽFig. 2E,F.. For the solitary nucleus ŽNTS., we analyzed three sections through the NTS at the level of the area postrema ŽFig. 2D,E., three sections rostral to the area postrema ŽFig. 2C. and three sections caudal to calamus ŽFig. 2F.. Separate cell counts were performed for the ventrolateral, dorsomedial, medial, and commissural NTS. For the Botzingerrpre-Botzinger ¨ ¨ complex w21,39x, three sections located just caudally to the facial nucleus were counted ŽFig. 2A.. The RVLM was analyzed in three sections rostral to the area postrema, according to Fig. 2B,C while the CVLM was analyzed in three sections according to Fig. 2E,F. For the A5-region, we counted three sections at the level of the superior olivary complex, according to figures 55–58 in Paxinos and Watson w34x. All values are expressed as mean " standard errors of the meanŽ"S.E.M... For statistical analysis of the differences between stimulation and control group we performed the analysis of variance ŽANOVA.. Probability values of p F 0.05 Ž). were considered to be significant Ž)) p F 0.01, ))) p F 0.001..
3.1. General distribution of Fos-ir neurons and NR1-r Fos-ir double-labeled neurons in pons and medulla Following nasal stimulation, we observed a distinct pattern of Fos-ir and NR1-rFos-ir double-labeled neurons in pontine and medullary nuclei. Fos-ir was prominent in the KF and in selected nuclei of the PB, such as the outer part of the external lateral PB, the central lateral PB and the superior lateral PB ŽFig. 1, Table 1.. The remaining PB nuclei showed only low numbers of Fos-ir neurons. Neurons double-labeled for Fos-ir and the NR1-subunit of the NMDA receptor were most numerous in the midlevel of the KF ŽFig. 1B,C and Fig. 3A. and in the superior lateral PB ŽFig. 1A. whereas the external lateral and central lateral PB exhibited considerably lower numbers of double-labeled cells ŽFig. 1B,C.. In the lower brainstem, many Fos-ir neurons were seen in the Botzingerrpre-Botzinger complex, in the rostral and ¨ ¨ caudal ventrolateral medulla, in the layers IrII of the Sp5C, and in the ventrolateral, dorsomedial, medial and commissural nuclei of the NTS ŽFig. 2, Table 2.. With respect to the numbers of Fos-rNR1-ir double-labeled neurons, differences were apparent between these nuclei. High ratios of double-labeled neurons were present in the Botzingerrpre-Botzinger complex ŽFig. 2A and Fig. 3C., ¨ ¨ along the rostrocaudal extend of the ventrolateral medulla ŽFigs. 2 and 3C,D., and in the Sp5C at the level of the area postrema and caudal to it ŽFig. 2E,F and Fig. 3B.. Except for the dorsomedial NTS, we found also considerable ratios of double-labeled neurons in the ventrolateral, medial and commissural NTS ŽFig. 2C–F.. 3.2. Statistical eÕaluation of Fos-ir neurons Compared to the control experiments, we observed that nasal stimulation evoked significant increases of Fos-positive neurons in selected pontine and medullary nuclei. In the KF, we found a highly significant increase of Fos-ir neurons in the midlevel KF Ž p s 0.0001. and a moderate increase in the rostral KF Ž p s 0.031.. In the PB, the numbers of Fos-ir neurons increased significantly in the outer external lateral PB Ž p s 0.0008. and in the central lateral PB Ž p s 0.015. while the superior lateral PB showed prominent numbers of Fos-ir cells in both, the stimulated and the control rats ŽTable 1.. In the medullary nuclei, we observed after nasal stimulation a highly significant increase of Fos-ir neurons in the Sp5C Ž p s 0.0003; Table 2.. Moderate but significant
Fig. 2. Camera lucida drawings of coronal sections through the medullary brainstem ŽA–F, from rostral to caudal. illustrating the distribution of Fos-ir Žfilled circles. and NR1-rFos-ir double-labeled Žopen squares. neurons in the ventrolateral medulla ŽBo, RVL, CVL., the solitary nucleus ŽNTS. and the spinal trigeminal nucleus ŽSp5C.. Sections shown in A–F correspond approximately to plates 65, 67, 70, 72, 74, 75, respectively of Paxinos and Watson w34x. Scale bar s 1 mm.
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Fig. 3. Photomicrographs of coronal sections through the Kolliker–Fuse nucleus ŽA., the spinal trigeminal nucleus ŽB., the rostral ventrolateral medulla at ¨ the level of the Botzinger complex ŽC., and the caudal ventrolateral medulla at the level of the area postrema ŽD. illustrating NR1-ir neurons Žfilled ¨ arrowheads., Fos-ir neurons Žopen arrowheads., and NR1-rFos-ir double-labeled neurons Žarrows.. Dorsal is to the top and lateral to the right in all photomicrographs. Scale bars s 20 mm in A, 50 mm in B–D.
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increases of Fos-ir neurons were also found in the Botzingerrpre-Botzinger complex Ž p s 0.032., in the ad¨ ¨ jacent rostral ventrolateral medulla ŽRVLM, p s 0.024. and in the ventrolateral Ž p s 0.022. and commissural NTS Ž p s 0.031; Table 2.. Remaining nuclei such as the medial and dorsomedial NTS showed prominent numbers of Fos-ir neurons, but no significant differences between stimulation and control ŽTable 2.. Furthermore, no significant differences in the numbers of Fos-ir neurons between stimulation and control was found in the caudal ventrolateral medulla and in the A5-region ŽTable 2.. 3.3. Statistical eÕaluation of NR1-rFos-ir double-labeled neurons Our immunocytochemical experiments revealed that all brain areas analyzed exhibit neurons double-labeled for NR1- and Fos-ir after both, nasal stimulation and anesthesia control. However, significant increases of NR1-rFos-ir double-labeled neurons after stimulation occurred only in a few brain areas. In the PBrKF complex, we found a significant increase in NR1-rFos-ir double-labeled neurons in the midlevel of the KF Ž p s 0.0011; Table 1. and a weak significance in the external lateral PB Ž p s 0.0435.. In the medullary nuclei, a highly significant increase in double-labeled cells could be observed in the Sp5C Ž p s 0.0002. while in the ventrolateral NTS Ž p s 0.0013. and in the Botzingerrpre-Botzinger complex Ž p s 0.048. the ¨ ¨ increase was moderately significant. In the remaining pontine and medullary nuclei, nasal stimulation did not result in a considerable increases of NR1-rFos-ir double-labeled cells ŽTables 1 and 2.. 4. Discussion 4.1. Technical considerations In the present study, we performed in the anesthetized rat noxious stimulation of the nasal mucosa which led to the expression of Fos-protein in the PBrKF, the A5-region, the NTS, the ventrolateral medulla, and the Sp5C. By means of immunocytochemical double-stainings, we revealed that in the brain areas analyzed considerable numbers of Fos-positive neurons also express NMDA receptors, as demonstrated by the presence of the NR1 subunit. In order to identify NMDA-type glutamate receptors, we used a monoclonal antibody that binds to the NR1 subunit w38x. The NR1 subunit is expressed abundantly in almost all regions of the rat brain w35x and is considered to be required in all functional NMDA receptors w19,42x. Therefore, the NR1 subunit probably serves as a valuable marker to localize NMDA receptors in rat brain tissue. Moreover, the antibody used is directed against a highly conserved loop of the NR1 protein and, thus, recognizes all of the presently known splice variants of the NR1 subunit w20,38x. The staining intensity for the NR1 subunit and, thus, the unequivocal detection of NR1-positive neurons is strongly
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dependent on the immunocytochemical procedure employed. Best staining results for NR1-ir were achieved when Triton X-100 was omitted from the incubation solutions. However, Triton X-100 treatment is required for the detection of the Fos protein. We therefore established an immunocytochemical staining protocol that allows for the sufficient detection of both antigens, Fos and NR1. Unfortunately, with this protocol the signal for NR1 became somewhat weaker compared to sections stained following the optimized NR1-protocol w15x. Occasionally, the lighter staining impeded an unequivocal decision whether or not a neuron exhibits NR1-ir. We therefore conclude that our cell counts most likely underestimate the real number of NR1-rFos-ir double-labeled neurons. As mentioned above, the pontine and medullary nuclei analyzed express remarkably high numbers of doublelabeled cells, presenting some evidence that Fos expression might depend on the presence of the NMDA receptor. The NMDA receptor is indeed a good candidate for being involved in the induction of the Fos protein since activation of NMDA receptors causes a strong influx of Ca2q-ions which were shown to be mandatory for Fos-induction w28x. Nevertheless, previous data on this topic are controversy with respect to the role of NMDA receptors for Fos-expression. It has been reported that injections of the NMDA receptor antagonist MK-801 strongly suppressed Fos expression in the Sp5C w4x while this was not the case with Fos expression in the spinal dorsal horn w41x. In the present study, we observed following nasal stimulation a significant increase of NR1-rFos-ir double-labeled cells only in selected brain nuclei whereas in others only the numbers of Fos-ir neurons increased significantly. Thus, it is conceivable to conclude that in those brain areas were we found significant increases in double-labeled cells, NMDA receptors are mandatory for both, Fos-expression and mediation of the cardiorespiratory reflex responses while this might not be the case in others. 4.2. NMDA receptors in mandatory and potential relay sites of the nasotrigeminal reflex circuit As demonstrated in the present study, repetitive noxious stimulation of the nasal mucosa induced a highly significant increase of NR1-rFos-ir double-labeled neurons in the midlevel KF and the Sp5C. These histological data suggest that neurotransmission in these mandatory relays for the nasotrigeminal reflex w9,10,32x is largely dependent on NMDA receptors. The KF, also known as the pontine pneumotaxic center w8x, represents the respiratory region of the parabrachial complex w6,11x. Our finding that in the midlevel of the KF a large population of neurons was double-labeled is in good agreement with recent pharmacological data from our laboratory. We observed that antagonism of NMDA receptors in the KF blocked the nasotrigeminally evoked apnea and bradycardia w11x while blockade of AMPA-type glutamate receptors was largely
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ineffective. An important role for NMDA receptors in the KF is also indicated by an autoradiographic binding study which revealed the highest levels of NMDA binding sites in the PBrKF, compared to other pontine regions w27x. In the Sp5C, the mediation of the nasotrigeminal reflex is apparently also dependent on glutamatergic neurotransmission w25x. Our data now indicate that it is the NMDA receptor that might play a crucial role for reflex mediation in the Sp5C, as it is the case for the KF. In the ventrolateral NTS, also referred to as the dorsal respiratory group, we found a significant increase of nasally induced Fos-ir and NR1-rFos-ir double-labeled neurons. This finding is in line with physiological investigations demonstrating the involvement of dorsal respiratory group neurons in mediating respiratory responses to trigeminal ethmoidal nerve stimulation w5x. NMDA receptors in ventrolateral NTS might participate in modulating the respiratory rhythm following nasotrigeminal stimulation. In the medial and dorsomedial NTS, the numbers of Fos-ir and NR1-rFos-ir double-labeled neurons were similar after both, nasal stimulation and anesthesia control. As discussed earlier w10x, the strong Fos expression in the NTS is most likely caused by blood pressure changes which also occur in response to the anesthetic alone. Nevertheless, we could recently demonstrate that the medial NTS at the level of the area postrema represents also a mandatory relay site for the nasotrigeminal reflex mediating selectively the arterial blood pressure response to nasal stimulation w12x. We speculate that trigeminal afferents, which in fact terminate in the NTS w2,26,33x, converge on NTS neurons that participate in blood pressure regulation w7x. The present data now show that part of the Fos-positive neurons also express the NR1 subunit. However, whether or not NMDA receptors participate in mediating the nasotrigeminal pressor response in the medial NTS needs to be tested pharmacologically. The elevated level of Fos-expression in the commissural NTS is in line with our previous report w10x. Nevertheless, a recent study revealed w12x that presynaptic blockade in the commissural NTS failed to block consistently the nasotrigeminally evoked autonomic responses. Thus, a possible role of the commissural NTS for mediating nasotrigeminal reflex responses as indicated by our reports remains to be demonstrated. In the ventrolateral medulla, there was a remarkably high ratio of NR1-rFos-ir double-labeled cells, particularly in the rostral part. A significant increase of both, Fos-ir and double labeled neurons occurred only in the region of the Botzingerrpre-Botzinger complex w21,39x ¨ ¨ while in the ventrolateral medulla caudally to the preBotzinger only the numbers of Fos-ir neurons had in¨ creased significantly. Our finding that nasal stimulation activates neurons in the ventral respiratory group is in accord with electrophysiological studies demonstrating a prominent drive of respiratory neurons in the ventral group after trigeminal ethmoidal nerve stimulation w3,40x. The fact that a large proportion of our Fos-positive neurons
express the NR1 subunit provides evidence that NMDA receptors might play a potent role in the modulation of the respiratory rhythm in response to nasal stimulation. In the lateral PB, the Fos-ir neurons were concentrated in the external lateral and central lateral PB which have long been known as major targets of NTS afferents w16x. As demonstrated in our previous report, linear correlations exist between the numbers of Fos-ir neurons in the NTS and the lateral PB, suggesting that the Fos-positive cells in the lateral PB are predominantly activated by NTS afferents w10x. To our knowledge, there is as yet no information about a potential role of these lateral PB nuclei and NMDA receptors in the nasotrigeminal reflex circuit. In the superior lateral PB, nasal stimulation with saline did not result in an increase of Fos-ir or NR1-rFos-ir doublelabeled neurons. Nevertheless, a high ratio of doublelabeled cells was present in the superior lateral PB in both, stimulated and control rats, indicating a potent role for NMDA receptors in this PB nucleus, which has been implicated in the processing of nociceptive information from various parts of the body w10,17,18x. 4.3. Conclusion Our data demonstrate that nasotrigeminally activated neurons in mandatory and potential relay sites of the nasotrigeminal reflex circuit express the NR1 subunit. This finding strongly suggests that NMDA-type glutamate receptors are involved in the mediation of the nasotrigeminally evoked cardiovascular and respiratory responses, which has been shown for the KF w11x. Pharmacological experiments are now needed to test this hypothesis for other brain areas as well.
5. List of abbreviations IrII 10 12 Amb AP Bar Bo Cu cu CVL DLL ECu GiV Gr IO KF lc LRt
Layers IrII of the spinal trigeminal nucleus, caudalis Dorsal motor nucleus of vagus Hypoglossal nucleus Ambiguus nucleus Area postrema Barrington’s nucleus Botzinger complex ¨ Cuneate nucleus Cuneate fasciculus Caudoventral reticular nucleus Dorsal nucleus lateral lemniscus External cuneate nucleus Gigantocellular reticular nucleus, ventralis Gracile nucleus Inferior olive Kolliker–Fuse nucleus ¨ Locus coeruleus Lateral reticular nucleus
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me5 Mo5 mlf MVe NR1 NTS PB Pr py R RVL scp SpVe sp5 Sp5C Sp5I Su5
Mesencephalic trigeminal tract Motor trigeminal nucleus Medial longitudinal fasciculus Medial vestibular nucleus NMDA receptor subunit 1 Nucleus tractus solitarius Parabrachial nucleus Prepositus nucleus Pyramidal tract Raphe nucleus Rostroventral reticular nucleus Superior cerebellar peduncle Spinal vestibular nucleus Spinal trigeminal tract Spinal trigeminal nucleus, caudalis Spinal trigeminal nucleus, interpolaris Supratrigeminal nucleus
Nuclei of the parabrachial complex c Central lateral PB d Dorsal lateral PB el External lateral PB el i Inner portion of the elPB el o Outer portion of the elPB exm External medial PB il Internal lateral PB m Medial PB s Superior lateral PB v Ventral lateral PB
Acknowledgements This study was supported by the Deutsche Forschungsgemeinschaft ŽHe1842r6-1. and by the Graduiertenkolleg Žstipend to M.D... The authors Neurobiologie Tubingen ¨ thank Helga Zillus for technical assistance with the histology and PD Dr. Michael Koch for valuable suggestions on the manuscript.
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Research report
NMDA receptor subunit NR1-immunoreactivity in the rat pons and brainstem and colocalization with Fos induced by nasal stimulation Mathias Dutschmann, Axel Guthmann, Horst Herbert
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Department of Animal Physiology, UniÕersity of Tubingen, Auf der Morgenstelle 28, D-72076 Tubingen, Germany ¨ ¨ Accepted 25 August 1998
Abstract In the present study, we examined the distribution of neurons in the parabrachial nucleus ŽPB., the Kolliker–Fuse nucleus ŽKF., the ¨ spinal trigeminal nucleus caudalis ŽSp5C., the nucleus of the solitary tract ŽNTS. and the ventrolateral medulla ŽVLM., which are activated by evoking the nasotrigeminal reflex and which exhibit immunoreactivity for the N-methyl-D-aspartate ŽNMDA. receptor subunit NR1. By stimulating the nasal mucosa with saline, we induced the expression of the immediate early gene c-fos and combined the immunocytochemical detection of the Fos protein with the detection of the NR1 subunit. Cell counts revealed that nasal stimulation, compared to anesthesia controls, resulted in highly significant increases Ž p F 0.001. of Fos-immunoreactive Ž-ir. neurons in the midlevel KF, the external lateral PB, and the Sp5C. In the central lateral PB, the rostral ventrolateral medulla including the Botzingerrpre-Botzinger ¨ ¨ complex, and in the ventrolateral and commissural NTS the increases were only moderately significant Ž p F 0.05.. With respect to the numbers of NR1-rFos-ir double-labeled neurons, significant increases were only observed in a subset of these pontomedullary nuclei. Increases were highly significant in the Sp5C Ž p F 0.001. and the midlevel KF Ž p F 0.01. and moderately significant Ž p F 0.05. in the external lateral PB, Botzingerrpre-Botzinger complex, and ventrolateral NTS. The present study revealed that nasotrigeminally activated ¨ ¨ neurons in mandatory and potential relay sites of the nasotrigeminal reflex circuit express the NR1 subunit. This finding strongly suggests that NMDA-type glutamate receptors are involved in the mediation of the nasotrigeminally evoked cardiovascular and respiratory responses. q 1998 Elsevier Science B.V. All rights reserved. Keywords: Parabrachial; Kolliker–Fuse; Solitarius; Trigeminal; Diving response; Sensory–autonomic integration ¨
1. Introduction Noxious stimulation of the nasal mucosa elicits a vital defensive reflex characterized by sudden cessation of breathing, bradycardia and peripheral vasoconstriction leading to a rise in arterial blood pressure w22x. These autonomic responses have been reported for several species w1,22,23,29x and are prominently expressed in diving mammals w13x. Sensory information from the nasal mucosa reaches the CNS via the ethmoidal nerve ŽEN5., a branch of the ophthalmic division of the trigeminal nerve w36,37x. Anatomical and pharmacological studies demonstrated that the first central relay site for EN5 afferents lies within the pars caudalis of the spinal trigeminal nucleus ŽSp5C. w2,24,25,30,32x. Furthermore, it was shown that the pontine ŽKF. nucleus respiratory neurons of the Kolliker–Fuse ¨ ) Corresponding author. Fax: q49-7071-292618; E-mail: [email protected]
w6,8x, which receive extensive projections from the Sp5C w14,31x, are also mandatory relays for the respiratory responses evoked by EN5 stimulation w9x. Most recently, we demonstrated that injections into the KF of the NMDA receptor antagonist AP5 blocked the EN5-evoked cardiorespiratory responses, indicating a major role for NMDA receptors in the mediation of the nasotrigeminal reflex w11x. In the present study, we investigated the potential involvement of NMDA receptors in other brain areas for mediating the nasotrigeminal reflex responses. By means of the stimulation triggered expression of the immediate early gene c-fos, we first identified neurons in pons and medulla that were activated after evoking the nasotrigeminal reflex w10x. We then stained the sections for the NMDA receptor-NR1 subunit to see if nasotrigeminally driven neurons also express NMDA receptors. Our findings may provide the basis for pharmacological experiments to further characterize the functional role of NMDA receptors
0006-8993r98r$ - see front matter q 1998 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 6 - 8 9 9 3 Ž 9 8 . 0 0 8 8 5 - 3
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Table 1 Summary of cell counts Žmean " S.E.M.. of Fos-ir neurons and NR1-rFos-ir double-labeled neurons in selected nuclei of the PBrKF complex after nasal stimulation with saline Ž n s 5. and after anesthesia control Ž n s 3. KF and PB nuclei
KF rostral aspects KF midlevel KF caudal aspects Superior lateral PB Central lateral PB External lateral PB
Fos-ir neurons
NR1-rFos-ir double-labeled neurons % Fos-ir neurons expressing NR1-ir
Stimulated
Control
Stimulated
Control
ŽStimulated group.
38 " 4) 239 " 16))) 33 " 7 49 " 5 104 " 16) 177 " 9)))
23 " 3 43 " 3 10 " 3 36 " 3 34 " 1 40 " 2
18 " 2 152 " 9)) 13 " 3 28 " 4 28 " 6 54 " 8)
16 " 2 23 " 5 8"3 23 " 3 20 " 1 25 " 1
47% 64% 34% 57% 27% 31%
for mediating the nasotrigeminal reflex response in different brain areas.
2. Materials and methods The investigations were carried out in accordance with the ethical guidelines for the care and use of experimental animals and were approved by the local council of animal care. We used male Wistar rats weighing 300–350 g which were housed under identical conditions in a room with a regular light–dark-cycle Ž06:00 h lights on, 18:00 h lights off. and food and water ad libitum. Rats used for the experiments were quickly removed from their home cages, anesthetized and then transferred to the laboratory. The data presented were derived from a total of eight rats in which the experimental manipulations and the immunocytochemical detection of the Fos protein and the NMDA receptor-NR1 subunit were successfully performed. Rats in which any step of the experimental protocol differed from the regular procedure were excluded from further analysis. 2.1. Experimental procedures To investigate the distribution of Fos- and NR1-rFosimmunoreactive Ž-ir. neurons in the pons and brainstem, we analyzed the brains from two experimental groups. In the first group, five rats were anesthetized with a mixture
of a-chloralose Ž150 mg kgy1 . and urethane Ž60 mg kgy1 . injected i.p. and placed on a heating pad Ž368C. to maintain body temperature. A small polyethylene tubing was inserted into the right nasal cavity and 0.2–0.3 ml saline at room temperature were infused. The saline infusion generally causes a pronounced suppression of breathing w10x. After 10 s, the saline was removed from the nasal cavity by suction. This stimulus was presented every 5 min for 1 h. The animals were killed 1.5 h after the last stimulation was given. The second group of rats Ž n s 3. served as controls. These rats were anesthetized as described above and then kept for 2.5 h on the heading pad in the surgery room. Thereafter, they were killed by transcardial perfusion and their brains were removed. For further details and extensive control experiments, see also Ref. w10x. 2.2. Tissue preparation At the end of the experiments, the stimulated rats were injected with a lethal dose of urethane Ž60 mg kgy1 . and transcardially perfused with 30 ml of phosphate buffered saline ŽPBS, 0.01 M at pH 7.4., followed by 500 ml of fixative containing 4% paraformaldehyde, 7.85 g lysine and 1.075 g sodium-meta-periodate in PBS. The brains were removed from the skull, postfixed in the same fixative for 1 h, placed in PBS containing 30% sucrose and stored at 48C. Coronal sections were cut at 40 mm on a freezing microtome and divided into three series. One
Table 2 Summary of cell counts Žmean " S.E.M.. of Fos-ir neurons and NR1-rFos-ir double-labeled neurons in selected nuclei of the medullary brainstem after nasal stimulation with saline Ž n s 5. and after anesthesia control Ž n s 3. Medullary nuclei
Sp5C A5-region Botzingerrpre-Botzinger ¨ ¨ RVLM CVLM Ventrolateral NTS Dorsomedial NTS Medial NTS Commissural NTS
Fos-ir neurons
NR1-rFos-ir double-labeled neurons % Fos-ir neurons expressing NR1-ir
Stimulated
Control
Stimulated
Control
ŽStimulated group.
261 " 40))) 24 " 3 40 " 7) 52 " 6) 41 " 5 109 " 21) 72 " 16 99 " 18 80 " 9)
5"1 27 " 6 12 " 2 24 " 3 31 " 12 30 " 19 68 " 14 104 " 21 46 " 5
139 " 18))) 13 " 2 32 " 8) 34 " 6 21 " 3 51 " 8) 16 " 3 41 " 9 33 " 4
3"1 18 " 6 8"2 20 " 4 25 " 12 17 " 11 17 " 4 43 " 14 25 " 1
53% 54% 80% 66% 52% 47% 21% 41% 41%
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series of sections was stained for Nissl-substance with thionine, the other two series were processed immunocytochemically. For immunocytochemistry, the sections were rinsed in Tris-buffered saline ŽTBS, 0.05 M at pH 7.6.. Nonspecific binding sites were blocked by incubating the sections in a solution containing 10% normal swine serum and 0.3% Triton X-100 in TBS. Thereafter, the sections were incubated in a sheep anti-Fos antiserum ŽCambridge Research Biochemicals, Northwich, UK. diluted 1:1000 in a carrier solution containing 2% normal swine serum and 0.3% Triton X-100 in TBS. After 48 h at 48C, the sections were rinsed in TBS and incubated for 1.5 h at room temperature in a biotinylated donkey anti-goat IgG antiserum ŽJackson ImmunoResearch, Dianova, Hamburg, Germany. diluted 1:1000 in carrier. Then, the sections were washed again in TBS and incubated for 1.5 h at room temperature in Avidin-Neutralite HRP ŽMolecular Probes Europe, Leiden, The Netherlands. diluted 1:1000 in carrier. After several rinses in TBS, the sections were incubated in 0.02% 3,3X-diaminobenzidine tetrahydrochloride ŽDAB; Sigma-Aldrich, Deisenhofen, Germany. in the presence of 0.01% H 2 O 2 and 0.3% Ni-ammonium sulfate ŽpH 8. for 10–30 min at room temperature, resulting in a black reaction product. The reaction was terminated by transferring the sections into TBS. For detection of the second antigen, the sections were washed again in TBS and incubated for 1 h in TBS containing 10% normal swine serum. Triton X-100 was omitted in all subsequent steps of the protocol. The sections were then incubated in a mouse anti-NMDA-NR1 antibody ŽPharMingen Deutschland, Hamburg, Germany.. This antibody is directed against a highly conserved amino acid sequence Ž660–811. and, thus, recognizes all isoforms of the NR1 subunit w38x. The sections were incubated in the primary antibody, diluted 1:200 in carrier containing 2% swine serum. After 48 h at 48C, the sections were rinsed in TBS and placed for 1 h at room temperature in a biotinylated goat anti-mouse IgG antiserum ŽJackson. diluted 1:200 in carrier solution, followed by repeated washes and incubation in a peroxidase-labeled avidin–biotin complex ŽABC, Vectastain Elite kit, Vector Laboratories, Burlingame, CA, USA.. Following rinses in TBS, the sections were incubated in 0.05% DAB in the presence of 0.01% H 2 O 2 for 10–30 min at room temperature, resulting in a brown reaction product. The reaction was terminated by transferring the sections in TBS. The sections were then mounted on gelatin-coated slides, air-dried, dehydrated in alcohols, cleared in xylene, and coverslipped with Entellan ŽMerck, Darmstadt, Germany.. Fig. 1. Camera lucida drawings of coronal sections through the dorsal pons ŽA–D, from rostral to caudal. illustrating the distribution of Fos-ir Žfilled circles. and NR1-rFos-ir double-labeled Žopen squares. neurons in the nuclei of the PBrKF complex. Sections shown in A–D correspond approximately to plates 53–56 of Paxinos and Watson w34x. Scale bar s 500 mm.
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2.3. Data analysis
3. Results
The sections were analyzed with a light microscope ŽReichert-Jung, Leica, Bensheim, Germany.. The distribution of Fos-ir neurons and neurons immunoreactive for Fos and NR1 protein ŽNR1-rFos-ir. was plotted with a camera lucida in the PBrKF nuclei and in the medullary brainstem. Cytoarchitectural boundaries of PBrKF and medullary nuclei were defined by superimposing the adjacent thionine-stained sections with the camera lucida drawings. The criteria for a neuron to be considered as doublelabeled were such that they had to exhibit a clearly blackstained cell nucleus and, in addition, a brown-stained cell body with clearly recognizable brown-stained primary dendrites. Representative examples of single and doublelabeled neurons were photographed. To evaluate the numbers of Fos-ir neurons and NR1-rFos-ir double-labeled neurons, we performed cell counts ipsilaterally in the brains of five stimulated rats and three control rats Žsummarized in Tables 1 and 2.. In brief, we counted cells in every third section through the pons and medulla. For the PBrKF, a total of eight sections through different rostrocaudal levels were analyzed: two sections from rostral levels Žcorresponding to the level shown in Fig. 1A., four sections from the midlevel ŽFig. 1B,C. and two sections from caudal levels ŽFig. 1D.. Separate cell counts were performed for the KF and the superior lateral, central lateral and external lateral PB. For the Sp5C, we analyzed three sections through the Sp5C at the level of the area postrema and just caudal to it ŽFig. 2E,F.. For the solitary nucleus ŽNTS., we analyzed three sections through the NTS at the level of the area postrema ŽFig. 2D,E., three sections rostral to the area postrema ŽFig. 2C. and three sections caudal to calamus ŽFig. 2F.. Separate cell counts were performed for the ventrolateral, dorsomedial, medial, and commissural NTS. For the Botzingerrpre-Botzinger ¨ ¨ complex w21,39x, three sections located just caudally to the facial nucleus were counted ŽFig. 2A.. The RVLM was analyzed in three sections rostral to the area postrema, according to Fig. 2B,C while the CVLM was analyzed in three sections according to Fig. 2E,F. For the A5-region, we counted three sections at the level of the superior olivary complex, according to figures 55–58 in Paxinos and Watson w34x. All values are expressed as mean " standard errors of the meanŽ"S.E.M... For statistical analysis of the differences between stimulation and control group we performed the analysis of variance ŽANOVA.. Probability values of p F 0.05 Ž). were considered to be significant Ž)) p F 0.01, ))) p F 0.001..
3.1. General distribution of Fos-ir neurons and NR1-r Fos-ir double-labeled neurons in pons and medulla Following nasal stimulation, we observed a distinct pattern of Fos-ir and NR1-rFos-ir double-labeled neurons in pontine and medullary nuclei. Fos-ir was prominent in the KF and in selected nuclei of the PB, such as the outer part of the external lateral PB, the central lateral PB and the superior lateral PB ŽFig. 1, Table 1.. The remaining PB nuclei showed only low numbers of Fos-ir neurons. Neurons double-labeled for Fos-ir and the NR1-subunit of the NMDA receptor were most numerous in the midlevel of the KF ŽFig. 1B,C and Fig. 3A. and in the superior lateral PB ŽFig. 1A. whereas the external lateral and central lateral PB exhibited considerably lower numbers of double-labeled cells ŽFig. 1B,C.. In the lower brainstem, many Fos-ir neurons were seen in the Botzingerrpre-Botzinger complex, in the rostral and ¨ ¨ caudal ventrolateral medulla, in the layers IrII of the Sp5C, and in the ventrolateral, dorsomedial, medial and commissural nuclei of the NTS ŽFig. 2, Table 2.. With respect to the numbers of Fos-rNR1-ir double-labeled neurons, differences were apparent between these nuclei. High ratios of double-labeled neurons were present in the Botzingerrpre-Botzinger complex ŽFig. 2A and Fig. 3C., ¨ ¨ along the rostrocaudal extend of the ventrolateral medulla ŽFigs. 2 and 3C,D., and in the Sp5C at the level of the area postrema and caudal to it ŽFig. 2E,F and Fig. 3B.. Except for the dorsomedial NTS, we found also considerable ratios of double-labeled neurons in the ventrolateral, medial and commissural NTS ŽFig. 2C–F.. 3.2. Statistical eÕaluation of Fos-ir neurons Compared to the control experiments, we observed that nasal stimulation evoked significant increases of Fos-positive neurons in selected pontine and medullary nuclei. In the KF, we found a highly significant increase of Fos-ir neurons in the midlevel KF Ž p s 0.0001. and a moderate increase in the rostral KF Ž p s 0.031.. In the PB, the numbers of Fos-ir neurons increased significantly in the outer external lateral PB Ž p s 0.0008. and in the central lateral PB Ž p s 0.015. while the superior lateral PB showed prominent numbers of Fos-ir cells in both, the stimulated and the control rats ŽTable 1.. In the medullary nuclei, we observed after nasal stimulation a highly significant increase of Fos-ir neurons in the Sp5C Ž p s 0.0003; Table 2.. Moderate but significant
Fig. 2. Camera lucida drawings of coronal sections through the medullary brainstem ŽA–F, from rostral to caudal. illustrating the distribution of Fos-ir Žfilled circles. and NR1-rFos-ir double-labeled Žopen squares. neurons in the ventrolateral medulla ŽBo, RVL, CVL., the solitary nucleus ŽNTS. and the spinal trigeminal nucleus ŽSp5C.. Sections shown in A–F correspond approximately to plates 65, 67, 70, 72, 74, 75, respectively of Paxinos and Watson w34x. Scale bar s 1 mm.
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Fig. 3. Photomicrographs of coronal sections through the Kolliker–Fuse nucleus ŽA., the spinal trigeminal nucleus ŽB., the rostral ventrolateral medulla at ¨ the level of the Botzinger complex ŽC., and the caudal ventrolateral medulla at the level of the area postrema ŽD. illustrating NR1-ir neurons Žfilled ¨ arrowheads., Fos-ir neurons Žopen arrowheads., and NR1-rFos-ir double-labeled neurons Žarrows.. Dorsal is to the top and lateral to the right in all photomicrographs. Scale bars s 20 mm in A, 50 mm in B–D.
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increases of Fos-ir neurons were also found in the Botzingerrpre-Botzinger complex Ž p s 0.032., in the ad¨ ¨ jacent rostral ventrolateral medulla ŽRVLM, p s 0.024. and in the ventrolateral Ž p s 0.022. and commissural NTS Ž p s 0.031; Table 2.. Remaining nuclei such as the medial and dorsomedial NTS showed prominent numbers of Fos-ir neurons, but no significant differences between stimulation and control ŽTable 2.. Furthermore, no significant differences in the numbers of Fos-ir neurons between stimulation and control was found in the caudal ventrolateral medulla and in the A5-region ŽTable 2.. 3.3. Statistical eÕaluation of NR1-rFos-ir double-labeled neurons Our immunocytochemical experiments revealed that all brain areas analyzed exhibit neurons double-labeled for NR1- and Fos-ir after both, nasal stimulation and anesthesia control. However, significant increases of NR1-rFos-ir double-labeled neurons after stimulation occurred only in a few brain areas. In the PBrKF complex, we found a significant increase in NR1-rFos-ir double-labeled neurons in the midlevel of the KF Ž p s 0.0011; Table 1. and a weak significance in the external lateral PB Ž p s 0.0435.. In the medullary nuclei, a highly significant increase in double-labeled cells could be observed in the Sp5C Ž p s 0.0002. while in the ventrolateral NTS Ž p s 0.0013. and in the Botzingerrpre-Botzinger complex Ž p s 0.048. the ¨ ¨ increase was moderately significant. In the remaining pontine and medullary nuclei, nasal stimulation did not result in a considerable increases of NR1-rFos-ir double-labeled cells ŽTables 1 and 2.. 4. Discussion 4.1. Technical considerations In the present study, we performed in the anesthetized rat noxious stimulation of the nasal mucosa which led to the expression of Fos-protein in the PBrKF, the A5-region, the NTS, the ventrolateral medulla, and the Sp5C. By means of immunocytochemical double-stainings, we revealed that in the brain areas analyzed considerable numbers of Fos-positive neurons also express NMDA receptors, as demonstrated by the presence of the NR1 subunit. In order to identify NMDA-type glutamate receptors, we used a monoclonal antibody that binds to the NR1 subunit w38x. The NR1 subunit is expressed abundantly in almost all regions of the rat brain w35x and is considered to be required in all functional NMDA receptors w19,42x. Therefore, the NR1 subunit probably serves as a valuable marker to localize NMDA receptors in rat brain tissue. Moreover, the antibody used is directed against a highly conserved loop of the NR1 protein and, thus, recognizes all of the presently known splice variants of the NR1 subunit w20,38x. The staining intensity for the NR1 subunit and, thus, the unequivocal detection of NR1-positive neurons is strongly
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dependent on the immunocytochemical procedure employed. Best staining results for NR1-ir were achieved when Triton X-100 was omitted from the incubation solutions. However, Triton X-100 treatment is required for the detection of the Fos protein. We therefore established an immunocytochemical staining protocol that allows for the sufficient detection of both antigens, Fos and NR1. Unfortunately, with this protocol the signal for NR1 became somewhat weaker compared to sections stained following the optimized NR1-protocol w15x. Occasionally, the lighter staining impeded an unequivocal decision whether or not a neuron exhibits NR1-ir. We therefore conclude that our cell counts most likely underestimate the real number of NR1-rFos-ir double-labeled neurons. As mentioned above, the pontine and medullary nuclei analyzed express remarkably high numbers of doublelabeled cells, presenting some evidence that Fos expression might depend on the presence of the NMDA receptor. The NMDA receptor is indeed a good candidate for being involved in the induction of the Fos protein since activation of NMDA receptors causes a strong influx of Ca2q-ions which were shown to be mandatory for Fos-induction w28x. Nevertheless, previous data on this topic are controversy with respect to the role of NMDA receptors for Fos-expression. It has been reported that injections of the NMDA receptor antagonist MK-801 strongly suppressed Fos expression in the Sp5C w4x while this was not the case with Fos expression in the spinal dorsal horn w41x. In the present study, we observed following nasal stimulation a significant increase of NR1-rFos-ir double-labeled cells only in selected brain nuclei whereas in others only the numbers of Fos-ir neurons increased significantly. Thus, it is conceivable to conclude that in those brain areas were we found significant increases in double-labeled cells, NMDA receptors are mandatory for both, Fos-expression and mediation of the cardiorespiratory reflex responses while this might not be the case in others. 4.2. NMDA receptors in mandatory and potential relay sites of the nasotrigeminal reflex circuit As demonstrated in the present study, repetitive noxious stimulation of the nasal mucosa induced a highly significant increase of NR1-rFos-ir double-labeled neurons in the midlevel KF and the Sp5C. These histological data suggest that neurotransmission in these mandatory relays for the nasotrigeminal reflex w9,10,32x is largely dependent on NMDA receptors. The KF, also known as the pontine pneumotaxic center w8x, represents the respiratory region of the parabrachial complex w6,11x. Our finding that in the midlevel of the KF a large population of neurons was double-labeled is in good agreement with recent pharmacological data from our laboratory. We observed that antagonism of NMDA receptors in the KF blocked the nasotrigeminally evoked apnea and bradycardia w11x while blockade of AMPA-type glutamate receptors was largely
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ineffective. An important role for NMDA receptors in the KF is also indicated by an autoradiographic binding study which revealed the highest levels of NMDA binding sites in the PBrKF, compared to other pontine regions w27x. In the Sp5C, the mediation of the nasotrigeminal reflex is apparently also dependent on glutamatergic neurotransmission w25x. Our data now indicate that it is the NMDA receptor that might play a crucial role for reflex mediation in the Sp5C, as it is the case for the KF. In the ventrolateral NTS, also referred to as the dorsal respiratory group, we found a significant increase of nasally induced Fos-ir and NR1-rFos-ir double-labeled neurons. This finding is in line with physiological investigations demonstrating the involvement of dorsal respiratory group neurons in mediating respiratory responses to trigeminal ethmoidal nerve stimulation w5x. NMDA receptors in ventrolateral NTS might participate in modulating the respiratory rhythm following nasotrigeminal stimulation. In the medial and dorsomedial NTS, the numbers of Fos-ir and NR1-rFos-ir double-labeled neurons were similar after both, nasal stimulation and anesthesia control. As discussed earlier w10x, the strong Fos expression in the NTS is most likely caused by blood pressure changes which also occur in response to the anesthetic alone. Nevertheless, we could recently demonstrate that the medial NTS at the level of the area postrema represents also a mandatory relay site for the nasotrigeminal reflex mediating selectively the arterial blood pressure response to nasal stimulation w12x. We speculate that trigeminal afferents, which in fact terminate in the NTS w2,26,33x, converge on NTS neurons that participate in blood pressure regulation w7x. The present data now show that part of the Fos-positive neurons also express the NR1 subunit. However, whether or not NMDA receptors participate in mediating the nasotrigeminal pressor response in the medial NTS needs to be tested pharmacologically. The elevated level of Fos-expression in the commissural NTS is in line with our previous report w10x. Nevertheless, a recent study revealed w12x that presynaptic blockade in the commissural NTS failed to block consistently the nasotrigeminally evoked autonomic responses. Thus, a possible role of the commissural NTS for mediating nasotrigeminal reflex responses as indicated by our reports remains to be demonstrated. In the ventrolateral medulla, there was a remarkably high ratio of NR1-rFos-ir double-labeled cells, particularly in the rostral part. A significant increase of both, Fos-ir and double labeled neurons occurred only in the region of the Botzingerrpre-Botzinger complex w21,39x ¨ ¨ while in the ventrolateral medulla caudally to the preBotzinger only the numbers of Fos-ir neurons had in¨ creased significantly. Our finding that nasal stimulation activates neurons in the ventral respiratory group is in accord with electrophysiological studies demonstrating a prominent drive of respiratory neurons in the ventral group after trigeminal ethmoidal nerve stimulation w3,40x. The fact that a large proportion of our Fos-positive neurons
express the NR1 subunit provides evidence that NMDA receptors might play a potent role in the modulation of the respiratory rhythm in response to nasal stimulation. In the lateral PB, the Fos-ir neurons were concentrated in the external lateral and central lateral PB which have long been known as major targets of NTS afferents w16x. As demonstrated in our previous report, linear correlations exist between the numbers of Fos-ir neurons in the NTS and the lateral PB, suggesting that the Fos-positive cells in the lateral PB are predominantly activated by NTS afferents w10x. To our knowledge, there is as yet no information about a potential role of these lateral PB nuclei and NMDA receptors in the nasotrigeminal reflex circuit. In the superior lateral PB, nasal stimulation with saline did not result in an increase of Fos-ir or NR1-rFos-ir doublelabeled neurons. Nevertheless, a high ratio of doublelabeled cells was present in the superior lateral PB in both, stimulated and control rats, indicating a potent role for NMDA receptors in this PB nucleus, which has been implicated in the processing of nociceptive information from various parts of the body w10,17,18x. 4.3. Conclusion Our data demonstrate that nasotrigeminally activated neurons in mandatory and potential relay sites of the nasotrigeminal reflex circuit express the NR1 subunit. This finding strongly suggests that NMDA-type glutamate receptors are involved in the mediation of the nasotrigeminally evoked cardiovascular and respiratory responses, which has been shown for the KF w11x. Pharmacological experiments are now needed to test this hypothesis for other brain areas as well.
5. List of abbreviations IrII 10 12 Amb AP Bar Bo Cu cu CVL DLL ECu GiV Gr IO KF lc LRt
Layers IrII of the spinal trigeminal nucleus, caudalis Dorsal motor nucleus of vagus Hypoglossal nucleus Ambiguus nucleus Area postrema Barrington’s nucleus Botzinger complex ¨ Cuneate nucleus Cuneate fasciculus Caudoventral reticular nucleus Dorsal nucleus lateral lemniscus External cuneate nucleus Gigantocellular reticular nucleus, ventralis Gracile nucleus Inferior olive Kolliker–Fuse nucleus ¨ Locus coeruleus Lateral reticular nucleus
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me5 Mo5 mlf MVe NR1 NTS PB Pr py R RVL scp SpVe sp5 Sp5C Sp5I Su5
Mesencephalic trigeminal tract Motor trigeminal nucleus Medial longitudinal fasciculus Medial vestibular nucleus NMDA receptor subunit 1 Nucleus tractus solitarius Parabrachial nucleus Prepositus nucleus Pyramidal tract Raphe nucleus Rostroventral reticular nucleus Superior cerebellar peduncle Spinal vestibular nucleus Spinal trigeminal tract Spinal trigeminal nucleus, caudalis Spinal trigeminal nucleus, interpolaris Supratrigeminal nucleus
Nuclei of the parabrachial complex c Central lateral PB d Dorsal lateral PB el External lateral PB el i Inner portion of the elPB el o Outer portion of the elPB exm External medial PB il Internal lateral PB m Medial PB s Superior lateral PB v Ventral lateral PB
Acknowledgements This study was supported by the Deutsche Forschungsgemeinschaft ŽHe1842r6-1. and by the Graduiertenkolleg Žstipend to M.D... The authors Neurobiologie Tubingen ¨ thank Helga Zillus for technical assistance with the histology and PD Dr. Michael Koch for valuable suggestions on the manuscript.
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