Nadph-diaphorase in the spinal trigeminal nucleus oralis and rostral solitary tract nucleus of rats

Neuroscience Vol. 61, No. 3, pp. 587-595, 1994

Pergamon

0306-4522(94)E0129-R

Elsevier ScienceLtd Copyright © 1994 IBRO Printed in Great Britain. All rights reserved 0306-4522/94 $7.00 + 0.00

NADPH-DIAPHORASE IN THE SPINAL TRIGEMINAL N U C L E U S ORALIS A N D ROSTRAL SOLITARY TRACT NUCLEUS OF RATS M. TAKEMURA,*tS. WAKISAKA,3~A. YOSHIDA,*Y. NAGASE,*Y. C. BAE§and Y. SHIGENAGA* Departments of Oral Anatomy (:~First and *Second Divisions), Osaka University Faculty of Dentristry, 1-8 Yamadaoka, Suita, Osaka 565, Japan §Department of Oral Anatomy, School of Dentristry, Kyungpook University, Taegu, Korea Al~traet--NADPH-diaphorase histochemical staining demonstrated a distinct neural group that might synthesize nitric oxide in the lower brainstem of rats. The NADPH-diaphorase stain revealed a Golgi-like network in the dorsomedial spinal trigeminal nucleus oralis and rostrolateral solitary tract nucleus, whereas this network was more dense in the latter nucleus. The distribution of NADPH-diaphorasepositive neurons in these areas overlapped with parts of central terminations from the chorda tympana nerve, as demonstrated with transganglionic transport of wheatgerm agglutinin conjugated horseradish peroxidase. The number of NADPH-diaphorase-positive neurons changed after chorda tympana nerve lesion relative to the contralateral side. The control value (%) was 106.0 + 4.9 (mean + S.E.M.). One hour after the nerve lesion, the value increased to 115.2 + 9.1 (P > 0.05). It then decreased to 83.9 ___5.2 two days after the lesion (P < 0.05), and remained at this reduced level for one or two weeks, 83.2 + 3.0 (P < 0.01) and 83.7 +2.3 (P < 0.01), respectively. This statistically significant reduction recovered to control level 103.4 + 2.9 four weeks after the lesion. These results show that NADPH-diaphorase-positive neurons in the lower brainstem could be regulated trans-synaptically by primary afferents, possibly gustatory inputs.

Sn might be regulated by primary afferent inputs from intraoral structures, 37'38 possibly gustatory input. The present study therefore aims to define the precise location of N A D P H - d neurons in the spinal trigeminai nucleus and Sn, and to examine the effects of chorda tympana nerve lesion on changes of N A D P H - d neurons.

Recently, nitric oxide (NO) has been shown to function as a neurotransmitter 5-7'36'42 with potential roles in long-term potentiation, plasticity, neurotoxicity u nociceptive processing, 2°,27'2s olfaction, 8 etc. In neurons, this highly toxic gas is produced by converting arginine to citrulline by the enzyme N O synthase (NOS) in a Ca 2 ÷/calmodulin-dependent manner. N O increases c G M P levels and augments the sensitivity of related receptors. Recent studies revealed that it may be possible to regard N A D P H - d i a p h o r a s e ( N A D P H d) a s N O S . 5'6'10'17 Exact mapping of the N A D P H d-positive neurons ( N A D P H - d neurons) in the C N S has been reported by many groups. 22m'43 A m o n g those reports, some investigators identified N A D P H d neurons in the spinal trigeminal nucleus 22 and solitary tract nucleus 43 (Sn). They demonstrated the presence of N A D P H - d neurons in these nuclei, but detailed characterization and the precise locations were not discussed. We have been interested in the functions of N A D P H - d neurons, and propose that those found in the spinal trigeminal nucleus and

EXPERIMENTAL PROCEDURES

Thirty male Sprague-Dawley rats (250-300 g body wt; Keari Co. Ltd, Osaka, Japan) were used. General anesthesia was induced using an intraperitoneal injection of sodium pentobarbital (25 mg/kg) and ethyl carbamate (600 mg/kg), and the chorda tympana nerve was lesioned unilaterally by abrasing the mucosa of the left tympana cavity with tweezers. We confirmed that this surgical technique resulted in complete lesion of the chorda tympanic nerve through the elimination of all central labeling of chorda tympana nerve (including all retrogradely labeled neurons in the superior salivatory nucleus) when horseradish peroxidase was applied to the lingual nerve. Labeling of the central trigeminal afferent terminals was not affected after this procedure. 37 The experimental animals were classified into five different groups in terms of postoperative periods of I h (n = 4), two days (n = 4), one week (n = 4), two weeks (n = 4) and four weeks (n = 5). Each animal was perfused intracardially with 100 ml saline followed by 500 ml of freshly prepared fixative containing 2.5% glutaraldehyde and 0.5% paraformaldehyde in 0.1 M phosphate buffer (PB; pH 7.4). 4~ Three animals with no surgical treatment were used to obtain parasagittal or horizontal sections of the lower brainstem, and four control animals were perfused identically for counting the NADPH-d neurons. The brainstem was postfixed in the same fixative for 2-24 h and stored in 20%

tTo whom correspondence should be addressed. dm, dorsomedial nucleus of Astrgm; NADPH-d, nicotinamide adenine dinucleotide phosphate-diaphorase; NO, nitric oxide; NOS, NO synthase; PB, phosphate buffer; PcRt, parvocellular reticular formation; Sn, solitary tract nucleus; SpVi, spinal trigeminal nucleus interpolaris; SpVo, spinal trigeminal nucleus oralis; T, trigeminal spinal tract; WGA-HRP, wheatgerm agglutinin conjugated horseradish peroxidase.

Abbreviations:

587

588

M. TAKEMURA et al.

sucrose in 0.1 M PB at 4°C until it sank for cryo-protection. Transverse, horizontal or parasagittal frozen sections were serially cut at 6 0 # m and collected in 0.1 M PB. For the visualization of the N A D P H - d we used nitro blue tetrazolium as chromogen and followed the published procedure. 4~ Sections were preincubated in 0.1 M PB containing 0.25% Triton X-100 for 5 min and incubated in a freshly prepared reaction solution containing 0.5 mg/ml B-NADPH (Oriental Yeast Co. Ltd, Osaka, Japan) and 0.2 mg/ml nitro blue tetrazolium (Sigma). The incubation was done at room temperature on a shaker for 3 5 h. Sections were rinsed in 0.1 M PB and distilled water, and then mounted on gelatincoated slides. Some sections were counterstained with Neutral Red. Postoperative animals were used for counting NADPH-d neurons in the spinal trigeminal nuleus oralis

4V 7M VIII CR DC EC f G

(SpVo) and rostral Sn. For quantitative analysis, every other transverse section was selected and the number of NADPHd neurons was counted. The ratios of N A D P H - d neurons in the lesioned (left) versus contralateral (right) sides were postoperatively determined. The statistical significance was assessed by Student's t-test. Two animals were used for demonstrating the central projection areas of chorda tympani nerve by using transganglionic transport of wheat germ agglutinin conjugated horseradish peroxidase (WGA-HRP). General anesthesia was induced using an intraperitoneal injection of ethyl carbamate (l.3 g/kg) and the lingual nerve was exposed by a submandibular skin incision and mylohyoid muscle retraction. The nerve was transected peripherally at the distal end of the bundle. In order to eliminate W G A - H R P transport

fourth ventricle facial motor nucleus vestibular root of the vestibulocochlear nerve corpus restiforme dorsal cochlear nucleus external cuneate nucleus facial nerve genu of the facial nerve

OS PV SSN St Tr V1 Vm Vsp

superior olive trigeminal sensory nucleus principalis superior salivatory nucleus solitary tract triangular-shaped tract of ,~str~m lateral vestibular nucleus medial vestibular nucleus spinal vestibular nucleus

Fig. 1.(a) Parasagittal section illustrating staining in the SpVo and Sn at low magnification. Large arrows indicate the levels of transverse sections illustrated in Fig. 3a-c arranged in rostrocaudal order. Arrowheads indicate the levels of horizontal sections demonstrated in Fig. 2a-c arranged in ventrodorsal order. Orientation arrows (d, dorsal; r, rostral) are 400 #m. (b-xl) Enlargements of N A D P H - d neural networks of the rostral (b), middle (c) and caudal (d) parts illustrated in a. Scale bar = 200 lam.

NADPH-diaphorase in the lower brainstem through the trigeminal nerve in experimental animals, the central trigeminal component was transected where the lingual nerve bifurcates into the trigeminal and facial components. Then, 1 gl 5% W G A - H R P (Sigma) entrapped in

589

2.8% hypoallergenic polyacrylamide geP 8 was injected into the lingual nerve with a 5.0 pl microsyringe. Before closing the incision, the surgical field was covered with petroleum jelly to prevent leakage of tracer into the surrounding

Fig. 2.(a-c) Horizontal sections illustrating staining in the SpVo and Sn at low magnification. Sections are arranged in ventrodorsal order. Arrowhead indicates the level of parasagittal section illustrated in Fig. 1. Rostrocaudal dimensions of a-c are arranged in the same position. Large arrows indicate the levels of transverse sections illustrated in Fig. 3a--c arranged in rostrocaudal order. Small arrows in c indicate NADPH-d axons that traverse T and triangular-shaped tract of Anstrom and join NADPH-d neural networks. Orientation arrows (m, medial; r, rostral) are 500/~m. (a'-c') Enlargements of NADPH-d neural networks in the same selections as a-c, respectively. Abbreviations as in Fig. 1. Scale bar = 200/~m.

590

M. TAKEMURAet al.

tissues. One day after the injection, animals were deeply anesthetized with ether and perfused intracardially with 100 ml of saline followed by 500 ml fixative containing 1% paraformaldehyde and 1.25% glutaraldehyde in 0.1 M PB and 500ml of 10% sucrose in 0.1 M PB at 4°C. Then the lower brainstem was postimmersed in 30% sucrose in 0.1 M PB at 4°C until it submerged. Sixty-micrometer transverse sections were made with a freezing microtome and incubated with tetramethylbenzidine according to Mesutam and Brushart?9 The labeled neuronal components were traced with the aid of a dark-field light microscope equipped with a camara lucida drawing tube. RESULTS

NADPH-diaphorase-positive neurons in the spinal trigeminal nucleus oralis and rostral solitary tract nucleus

In all animals, N A D P H - d neurons formed two distinct neuronal populations; one was in the dorsomedial SpVo and the other in the rostrolateral Sn (Figs la, 2a-c, 3a-c). The dorsomedial parts of the SpVo have been cytoarchitecturally subdivided into two groups, namely the rostro-dorso-medial part (see Torvick 39) and the dorsomedial nucleus (dm), as described by ,~,str~m. 2 The Sn appeared at the rostralmost level of the dm and was located just dorsomedial to it (Fig. 4c). The NADPH-d neurons were characterized by their formazan deposits in soma, dendrites and axon, except for the nucleus. Their somata were triangular, piriform or fusiform in shape (Figs l b ~ , 2a'-c'), with diameters ranging from 10 to 20 #m, but the cell size of SpVo neurons tended to be larger than that of Sn neurons. Dendrites of N A D P H - d neurons were generally long and straight with few branches (Figs lb-d, 2a'-c'). These morphological features of NADPH-d neurons were common to the neurons in SpVo and Sn, but patterns of cell arrangement and dendritic arborization differed between both nuclei. In SpVo, NADPH-d neurons were arranged in sporadic fashion (Figs lb-d, 2a', 3a-c). Their dendrites were generally emitted from the dorsal and ventral aspects of the soma and extended dorsally and ventrally, respectively (Fig. lb). The neurons situated more dorsally in the dm issued dendrites that oriented rostrocaudally (Figs lc, d, 2a'). It is noteworthy that dendrites could be followed to extend into the lateral Sn (see Discussion). For example, Sn received dendritic extension from dorsally and rostrocaudally oriented dendrites for ventrally and dorsally located SpVo neurons, respectively (Fig. lb-d). In Sn, on the other hand, N A D P H - d neurons formed a more dense dendritic plexus in its lateral parts (Fig. 3b, c). Rostrocaudally, the labeled neurons were seen at the rostralmost Sn, but they decreased in number at more caudal levels (Figs lc, d, 2b', c', 3b, c). Most neurons were distributed in a small area adjacent to the dm and issued stem dendrites from the caudal aspect of the soma (Fig. 2b', c'). Their dendrites extended caudomedially and formed a dense plexus of dendritic arbors (Fig. 2b',

c'). Consequently, the caudal regions of the Sn were characterized by dense dentritic arbors arising from neurons in its rostral regions, with less frequent distribution of somata (Figs ld, 2c', 3c). It is noteworthy that dendritic arbors originating in SpVo neurons participated in a dense dendritic plexus in the Sn, as mentioned previously. More caudally, at the level of the trigeminal nucleus interpolaris (SpVi), NADPH-d neurons also appeared in the medial Sn (Fig. 2C), where NADPHd-positive axon terminals possibly originating from vagus and glossopharyngeal nerves (Fig. 2c, small arrows) were observed, but neurons in the lateral Sn and dm were free from labeling. Consequently, we considered that the above-described NADPH-d neuron group was interrupted caudally at the transition of SpVo and SpVi, and that NADPH-d neurons in the caudal regions of the medial Sn might belong to a morphologically or functionally distinct neural group (data not shown). Finally, we observed another cell group positive for NADPH-d which constituted large multipolar cells in the parvocellular reticular formation (PcRt) at the rostral levels of the SpVo (Figs 2a, b, 3b). Their dendritic trees were mostly limited within the PcRt. Neurons in the superior salivatory nucleus and afferent component of the chorda tympani and trigeminal nerves were negative for NADPH-d stain. Centrally labeled chorda tympani nerve

Axon terminals labeled transganglionically and neurons retrogradely with W G A - H R P were recognized in the Sn and the superior salivatory nucleus (SSN in Fig. 4), respectively. In the rostralmost level of the SpVo, two distinct bundles of W G A - H R P labeled fibers were seen in the regions dorsal and ventral to the spinal trigeminal tract (T) (Fig. 4a, b). The ventral bundle directed dorsomedially along the medial SpVo to the facial genu, where it took a hairpin curve and finally reached the superior salivatory nucleus containing WGA-HRP-Iabeled multipolar neurons (Fig. 4a-c). The dorsal bundle traversed the T and dorsal SpVo mediodorsally, and joined the solitary tract (Fig. 4a, b). More caudally, the labeled bundle formed a compact terminal arbor between the dm and the labeled solitary tract (Fig. 4c). The rostralmost Sn is thus defined by the appearance of labeled terminals from the chorda tympani nerve, and it corresponds to the level of the rostralmost dm. Caudal sections revealed that the labeled terminal area in the Sn expanded medially, though its ventrolateral and dorsomedial areas were less labeled (Fig. 4d). At the SpVi level, the Sn was devoid of labeling. NADPH-diaphorase-positive neurons after chorda tympani nerve lesion

We could not detect any morphological changes, except for numbers of NADPH-d neurons, in the SpVo and Sn after chorda tympani nerve lesion,

NADPH-diaphorase in the lower brainstem compared to the contralateral side or control animals. Although N A D P H - d neurons formed a distinct dense neuronal plexus in the SpVo and Sn, the data were combined since there were no significant differ-

591

ences between the two nuclei (data not shown). In control animals (n = 4), the number of N A D P H - d neurons in the SpVo and Sn ranged from 206 to 400 on the ipsilateral side ( m e a n _ + S . E . M . = 342.3 + 45.8) and 209 to 402 in the contralateral side (mean + S.E.M. = 322.5 _ 42.3). The ratios of positive neurons in the Sn and SpVo of ipsilateral versus contralateral sides ranged from 98.6% to 119.7%. The mean + S.E.M. of the ratios was 106.0 ___4.9% (Fig. 5). The ratios of N A D P H - d neurons in the lesioned versus non-lesioned/contralateral side changed postoperatively (Fig. 5). One hour after the chorda tympani nerve lesion, there was a trend towards an increase in the ratio to 115.2 + 9.1%; this difference was not statistically significant. Two days after the denervation, the ratio decreased significantly to 83.9 _+ 5.2% (Fig. 5, ~t', P < 0.05). The value maintained a statistically significant decrease one and two weeks after the denervation at the ratio of 83.2 _ 3.0% ('k'~', P < 0.01) and 83.7 ± 2.3% ( . ~ ¢ , P < 0.01), respectively). Four weeks after the nerve lesion, the ratio returned to 103.4 __ 2.9%, essentially the control value. DISCUSSION

N A D P H - d neurons have been reported to be identical to neurons containing a soluble NOS by many investigators. 5'm'17'25 Matsumoto e t al. 25 argued that the coincidental correlation could be due to the inactivation of most non-NOS N A D P H - d activity during the fixation procedure. To address this concern in our preliminary study, we employed a higher concentration of fixative containing 4% paraformaldehyde or a longer period of postfixation (up to seven days) and were able to demonstrate substantial N A D P H - d staining. The location of N A D P H - d neurons in the SpVo and Sn appeared unaltered; however, the background and intensity of the labeling was somewhat attenuated. We demonstrate a distinct population of N A D P H d neurons, which are likely to release NO, in the SpVo and Sn, and that activity of chorda tympani afferents regulate these neurons trans-synaptically. The rostrolateral subdivision of the Sn is well known as a gustatory zone which receives projections not only from the chorda tympani, greater superficial petrosal and glossopharyngeal afferents, ~5 but also from trigeminal primary afferents innervating intraoral structures, e.g. the tongue, tooth and mucous

Fig. 3.(a-c) Transverse sections illustrating staining in the SpVo and Sn. Sections are arranged in rostrocaudal order. Mediolateral dimensions of all sections are arranged in the same position. Arrow indicates the position of parasagittal section illustrated in Fig. 1. Arrowheads indicate the levels of horizontal sections illustrated in Fig. 2 arranged in ventrodorsal order. Abbreviations as in Fig. 1. Orientation arrows (d, dorsal; 1, lateral) are 250 pm.

592

M. TAKEMURA el a].

SSN

~

+2.4

b _~_~~, ~v~

d

a-'-

$n

d

Fig. 4.(a-d) WGA-HRP-Iabeled central components of the chorda tympani nerve. Sections are arranged in rostrocaudal order. Numbers to the right of the figure represent the distance in millimeters rostral to the obex. Abbreviations as in Fig. 1. Orientation arrows (d, dorsal; I, lateral) are 400 #m.

NADPH-diaphorase in the lower brainstem

593

(~) 120 -

2

¢lJJ

~

°°°°°°~ o° °°°°° °°°°° o°°°

100

mQ. 0 ¢Q n-

I ° ° ° ° ° o ° y° ° °o°°°°oo°°°°oo°°° ° ° o ° ° ° ° °T° °°°'° 80

!

1

!

!

C lhr

!

2d

7d

14d

! 28d (Postoperative Time)

Fig. 5. Postoperative changes of NADPH-d neurons in the SpVo and Sn. St'Significantly different from control value at P < 0.05, -A"~P < 0.01.

membrane of cheek and palatinefl4'37'3s Our present results with W G A - H R P tracing method indicating the central terminal area and trajectory of chorda tympani nerve are consistent with a previous report.15 The terminal area of chorda tympani nerve overlapped with the territory of N A D P H - d neurons in the Sn, and with parts of the dendritic field of N A D P H - d neurons in the dorsomediai corner of the SpVo and dm. The latter two subdivisions receive projections from primary afferents innervating intra-oral structures only. 15'24'37'3sIn any case, N A D P H - d neurons in the SpVo and Sn receive inputs from both trigeminal and taste nerves. The N A D P H - d neurons in the Sn are supposed to be implicated in sensory processing of the intra-oral structures. On the other hand, the N A D P H - d neurons in rostral SpVo and dm are implicated in sensorimotor reflexes and in modulation of sensory discrimination, 13 because axons of nociceptive and mechanoreceptive neurons in these subdivisions terminate in the trigeminal motor nucleus, the sensory trigeminal nuclei and the PcRt, 1s'35 but few terminate in the nucleus ventralis posteromedialis of the thalamus. 14,34 The levels of certain proteins are regulated by altering the population of neurons expressing the protein in a regional fashion. 3°-3z.6 The increase of ratios of NADPH-d neurons observed 1 h after chorda tympani nerve lesion may be explained by the fact that transected primaries show an injury discharge45 and this unusual hyperactivity may cause trans-synaptic fluctuating up-regulation of enzyme NADPH-d. On the other hand, a decrease of N A D P H - d neurons was observed during the period from two days to two weeks after nerve lesion. This phenomenon will be evaluated from prior experimental studies. For example, neuropeptide y,44 vasoactive intestinal peptide and galanin have been reported to NSC 6 1 / 3 ~

increase their immunoreactivity in the medullary or spinal dorsal horns following peripheral nerve lesion. 3'16'26'3°'31'33On the contrary, levels of fluorideresistant acid phosphatase, 12substance p4,19 and caicitonin gene-related peptide 32 have been reported to decrease. In the SpVo and Sn, a dramatic increase in immunoreactivity of neuropeptide Y is observed in the central terminals of the inferior alveolar nerve after the nerve lesion has been made peripherally (Wakisaka et al., unpublished observations). Consequently, there is a possibility that some of the abovedescribed neuroactive substances may regulate trans-synaptically the level of NADPH-d of SpVo and Sn neurons. One line of evidence shows a transsynaptic regulation of NADPH-d activity in the lumbar spinal cord following unilateral hindpaw inflammation. 4° To our knowledge, the work we present here is the first to demonstrate the transsynaptic regulation of NADPH-d neurons in the SpVo and Sn by primary afferent lesion. In the present study, four weeks after the nerve lesion the ratio recovered to the control level. This is supported by prior studies4,'2 showing that substance P or other neuroactive peptides in primaries recover their activities after nerve lesion, relevant to nerve regeneration. Our experimental conditions do not address whether lesioning of chorda tympani nerve by abrasing mucosa of the tympanic cavity allows animals to regenerate within four weeks. We predict that there is some compensatory circuit which may help to regain the N A D P H - d activity in the SpVo and Sn within four weeks. Changes in the numbers of N A D P H - d neurons were observed not only in the Sn but also in the SpVo following nerve lesion, suggesting that dendrites of NADPH-d neurons in the SpVo receive direct input from chorda tympani afferents. The synaptic contacts between the two

594

M. TAKEMURAet al.

n e u r o n groups might be made on the dendrites extending into the Sn, because c h o r d a tympani afterents did not terminate in the SpVo. These findings also s u p p o r t o u r suggestion that N A D P H - d n e u r o n s observed in the SpVo a n d the lateral parts of rostral Sn belong to a morphologically h o m o l o g o u s n e u r o n group. F u r t h e r m o r e , the present findings that central processes of glossopharyngeal a n d vagal nerves are positive for N A D P H - d but those of c h o r d a tympani and trigeminal nerves are free from labeling, suggest that N A D P H - d is expressed in general visceral afferents 1 but not in general somatic and special visceral (taste) afferents. The functional role of N O in this area is largely u n k n o w n . In the dorsal horn, for example, N O has been implicated in nociceptive processing in a model of n e u r o p a t h i c pain t h r o u g h N-methyl-D-aspartate receptor activation. 27'28 We suppose that N O production in the Sn a n d SpVo is facilitated by the special visceral a n d general somatic afferent activities, a n d m a y be responsible for m o d u l a t i o n of reflexive s e n s o r i m o t o r functions and sensory discrimination.

There is little i n f o r m a t i o n a b o u t neuroactive substances in the Sn gustatory zone a n d the d m of the SpVo. Some groups reported that G A B A and G A B A - t r a n s a m i n a s e - p o s i t i v e neurons are present in the Sn gustatory zone. 2t G l u t a m a t e r g i c axons and neurons are distributed t h r o u g h o u t the spinal trigeminal nucleus. 9'23The relationship between these neuroactive substances a n d N A D P H - d neurons in this area and neural connections of the N A D P H - d neurons with other areas will be a future theme of our work.

CONCLUSIONS The present study, indicating changes of N A D P H d neurons in the SpVo a n d Sn after c h o r d a tympani nerve lesion, suggests that N O p r o d u c t i o n in these neurons is regulated trans-synaptically by special visceral and general somatic afferent activities. are indebted to Drs Takashi Yamamoto and David M. Donovan for valuable comments and discussion, and Ms Tomoko Koyama for excellent technical and secretarial assistance. Acknowledgements--We

REFERENCES

1. Aimi Y., Fujimura M., Vincent S. R. and Kimura H. (1991) Localization of NADPH-diaphorase-containing neurons in sensory ganglia of the rat. J. comp. Neurol. 306, 382-392. 2. /kstrom K. E. (1953) On the central course of afferent fibers in the trigeminal, facial, glossopharyngeal, and vagal nerves and their nuclei in the mouse. Acta physiol, scand. 29, Suppl. 106, 209 320. 3. Atkinson M. E. and Shehab S. A. S. (1986) Peripheral axotomy of the rat mandibular trigeminal nerve leads to an increase in VIP and decrease of other primary afferent neuropeptides in the spinal trigeminal nucleus. Regul. Pept. 16, 69-82. 4. Barbut D., Polak J. M. and Wall P. D. (1981) Substance P in spinal cord dorsal horn decreases following peripheral nerve injury. Brain Res. 205, 289--298. 5. Bredt D. S., Glatt C, E., Hwang P. M., Fotuhi M., Dawson T. M. and Snyder S. H. (1991) Nitric oxide synthase protein and mRNA are discretely localized in neuronal populations of the mammalian CNS together with NADPH diaphorase. Neuron 7, 615~24. 6. Bredt D. S., Hwang P. M. and Snyder S. H. (1990) Localization of nitric oxide synthase indicating a neural role for nitric oxide. Nature 347, 768-770. 7. Bredt D. S. and Snyder S. H. (1992) Nitric oxide, a novel neuronal messenger. Neuron 8, 3 11. 8. Breer H. and Shepherd G. M. (1993) Implications of the NO/cGMP system for olfaction. Trends Neurosci. 16, 5-9. 9. Clements J. R. and Beitz A. J . (1991) An electron microscopic description of glutamate-like immunoreactive axon terminals in the rat principal sensory and spinal trigeminal nuclei. J. comp. Neurot. 309, 271-280. 10. Dawson T. M., Bredt D. S., Fotuhi M., Hwang P. M. and Snyder S. H. (1991) Nitric oxide synthase and neuronal NADPH diaphorase are identical in brain and peripheral tissues. Proc. natn. Acad. Sci. U.S.A. 88, 7797 7801. I 1. Dawson V. L., Dawson T. M., London E. D., Bredt D. S. and Snyder S. H. (1991) Nitric oxide mediates glutamate neurotoxicity in primary cortical cultures. Proc. natn. Acad. Sci. U.S.A. 88, 6368~5371. 12. Devor M. and Claman D. (1980) Mapping and plasticity of acid phosphatase afferents in the rat dorsal horn. Brain Res. 190, 17-28. 13. Dubner R., Sessle B. J. and Storey A. T. (1978) The Neural Basis of Oral and Facial Function. Plenum Press, New York. 14. Fukushima T. and Kerr F. W. L. (1979) Organization of trigeminothalamic tracts and other thalamic afferent systems of the brainstem in the rat: presence of gelatinosa neurons with thalamic connections. J. comp. Neurol. 183, 169-184. 15. Hamilton R. B. and Norgren R. (1984) Central projections of gustatory nerves in the rat. J. comp. Neurol. 222, 560-577. 16. H6kfelt T., Wiesenfeld-Hallin Z., Villar M. and Melander T. (1987) Increase of galanin-like immunoreactivity in rat dorsal ganglion cells after peripheral axotomy. Neurosci. Lett. 83, 217~20. 17. Hope B. T., Michael G. J., Knigge K. M. and Vincent S. R. (1991) Neuronal NADPH diaphorase is a nitric oxide synthase. Proc. natn. Acad. Sci. U.S.A. 88, 2811-2814. 18. Jacquin M. F. and Rhoades R. W. (1986) Cell structure and response properties in the trigeminal subnucleus oralis. Somatosens. Motor Res. 61, 457468. 19. Jessell T., Tsunoo A., Kanazawa I. and Otsuka M. (1979) Substance P depletion in the dorsal horn of rat spinal cord after section of the peripheral processes of primary sensory neurons. Brain Res. 168, 247-259~ 20. Kitto K. F., Haley J. E. and Wilcox G. L. (1992) Involvement of nitric oxide in spinally mediated hyperalgesia in the mouse. Neurosci. Lett. 148, 1-5. 21. Lasiter P. S. and Kachele D. L. (1988) Organization of GABA and GABA-transaminase containing neurons in the gustatory zone of the nucleus of the solitary tract. Brain Res. Bull. 21, 623~536.

NADPH-diaphorase in the lower brainstem

595

22. Leigh P. N., Connick J. H. and Stone T. W. (1990) Distribution of NADPH-diaphorase positive cells in the rat brain. Comp. Biochem. Physiol. 97C, 259-264. 23. Magnusson K. R., Larson A. A., Madl J. E., Altschuler R. A. and Beitz A. J. (1986) Co-localization of fixative-modified glutamate and glutaminase in neurons of the spinal trigeminal nucleus of the rat: an immunohistochemical and immunoradiochemical analysis. J. comp. Neurol. 247, 477~490. 24. Marfurt C. F. and Rajchert D. M. (1991) Trigeminal primary afferent projections to "non-trigeminal" areas of the rat central nervous system. J. comp. Neurol. 303, 489-511. 25. Matsumoto T., Nakane M., Pollock J. S., Euk J. E. and Forstermann U. (1993) A correlation between soluble brain nitric oxide synthase and NADPH-diaphorase activity is only seen after exposure of the tissue to fixative. Neurosci. Lett. 155, 61~4. 26. McGregor G. P., Gibson S. J., Sabate I. M., Blank M. A., Christofides N. D., Wall P. D., Polak J. M. and Bloom S. R. (1984) Effect of peripheral nerve section and nerve crush on spinal cord neuropeptides in the rat; increased VIP and PHI in the dorsal horn. Neuroscience 13, 207-216. 27. Meller S. T. and Gebhart G. F. (1993) Nitric oxide (NO) and nociceptive processing in the spinal cord. Pain 52, 127-136. 28. Meller S. T., Pechman P. S., Gebhart G. F. and Maves T. J. (1992) Nitric oxide mediates the thermal hyperalgesia produced in a model of neuropathic pain in the rat. Neuroscience 50, 7-10. 29. Mesulam M. M. and Brushart T. M. (1979) Transganglionic and anterograde transport of horseradish peroxidase across dorsal root ganglia: a tetramethylbenzidine method for tracing central sensory connections of muscles and peripheral nerves. Neuroscience 4, 1107-1117. 30. Nielsch U. and Keen P. (1989) Reciprocal regulation of tachykinin- and vasoactive intestinal polypeptide-gene expression in rat sensory neurones following cut and crush injury. Brain Res. 481, 25-30. 31. Noguchi K., Senba E., Morita Y., Sato M. and Tohyama M. (1989) Prepro-VIP and preprotachykinin mRNAs in the rat dorsal root ganglion cells following peripheral axotomy. Molec. Brain Res. 6, 327-330. 32. Noguchi K., Senba E., Morita Y., Sato M. and Tohyama M. (1990) a-CGRP and fl-CGRP mRNAs are differentially regulated in the rat spinal cord and dorsal root ganglion. Molec. Brain Res. 7, 299-304. 33. Shehab S. A. S. and Atkinson M. E. (1986) Vasoactive intestinal polypeptide increases in areas of the dorsal horn of the spinal cord from which other neuropeptides are depleted following peripheral axotomy. Expl Brain Res. 62, 422430. 34. 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. 35. Shigenaga Y., Yoshida A., Mitsuhiro Y., Tsuru K. and Doe K. (1988) Morphological and functional properties of trigeminal nucleus oralis neurons projecting to the trigeminal motor nucleus of the cat. Brain Res. 461, 143-149. 36. Snyder S. H. (1992) Nitric oxide: first in a new class of neurotransmitters? Science 257, 494-496. 37. Takemura M., Sugimoto T. and Sakai A. (1987) Topographic organization of central terminal region of different sensory branches of the rat mandibular nerve. Expl Neurol. 96, 54(~557. 38. Takemura M., Sugimoto T. and Shigenaga Y. (1991) Difference in central projection of primary afferents innervating facial and intraoral structures in the rat. Expl Neurol. 111, 324 331. 39. Torvick A. (1956) Afferent connections to the sensory trigeminal nuclei, the nucleus of the solitary tract and adjacent structures: an experimental study in the rat. J. comp. Neurol. 106, 51-141. 40. Traub R. J., Solodkin A. and Gebhart G. F. (1992) A bilateral increase in NADPH-diaphorase activity in the rat lumbar spinal cord following unilateral hindpaw inflammation. Soc. Neurosci. Abstr. 19, 135. 41. Valtschanoff J. G., Weinberg R. J. and Rustioni A. (1992) NADPH diaphorase in the spinal cord of rats. J. comp. Neurol. 321, 209-222. 42. Vincent S. R. and Hope B. T. (1992) Neurons that say NO. Trends Neurosci. 15, 108-113. 43. Vincent S. R. and Kimura H. (1992) Histochemical mapping of nitric oxide synthase in the rat brain. Neuroscience 46, 755-784. 44. Wakisaka S., Kajander K. C. and Bennett G. J. (1991) Increased neuropeptide Y (NPY)-like immunoreactivity in rat sensory neurons following peripheral axotomy. Neurosci. Lett. 12,4, 200-203. 45. Wall P. D., Waxman S. and Basbaum A. I. (1974) Ongoing activity in peripheral nerve: injury discharge. Expl Neurol. 45, 576-589. 46. Walther D., Takemura M. and Uhl G. R. (1993) Fos family member changes in nucleus caudalis neurons after primary afferent stimulation: enhancement of fos B and c-fos. Molec. Brain Res. 17, 155-159. (Accepted 15 February 1994)