BRAIN RESEARCH

ELSEVIER Brain Research 735 (1996) 325-329

S h o r t c o m m u n i c a t i o n

Coexistence of calbindin D-28k and NADPH-diaphorase in vagal and 1 glossopharyngeal sensory neurons of the rat

H i r o y u k i I c h i k a w a a, C i n d a J. H e l k e b,,

a Second Department of Oral Anatomy, Okayama University Dental School, Okayama 700, Japan b Department of Pharmacology, Uniformed Services University of the Health Science, 4301 Jones Bridge Road, Bethesda, MD 20814, USA

Accepted 2 July 1996

Abstract

The presence and coexistence of calbindin D-28k-immunoreactivity (ir) and nicotinamide adenosine dinucleotide phosphate (NADPH)-diaphorase activity (a marker of neurons that are presumed to convert L-arginine to L-citrulline and nitric oxide) were examined in the glossopharyngeal and vagal sensory ganglia (jugular, petrosal and nodose ganglia) of the rat. Calbindin D-28k-ir nerve cells were found in moderate and large numbers in the petrosal and nodose ganglia, respectively. Some calbindin D-28k-ir nerve cells were also observed in the jugular ganglion. NADPH-diaphorase positive nerve cells were localized to the jugular and nodose ganglia and were rare in the petrosal ganglion. A considerable portion (33-51%) of the NADPH-diaphorase positive neurons in these ganglia colocalized calbindin D-28k-ir. The presence and colocalization of calbindin D-28k-ir and NADPH-diaphorase activity in neurotransmitter-identified subpopulations of visceral sensory neurons were also studied. In all three ganglia, calcitonin gene-related peptide (CGRP)-ir was present in many NADPH-diaphorase positive neurons, a subset of which also contained calbindin D-28k-ir. In the nodose ganglion, many (42%) of tyrosine hydroxylase (TH)-ir neurons also contained NADPH diaphorase activity but did not contain calbindin D-28k-ir. These data are consistent with a potential co-operative role for calbindin D-28k and NADPH-diaphorase in the functions of a subpopulation of vagal and glossopharyngeal sensory neurons.

Keywords: Calbindin D-28k; Calcitonin gene-related peptide; Immunohistochemistry; Jugular ganglion; NADPH-diaphorase; Nodose ganglion; Petrosal ganglion; Tyrosine hydroxylase

Sensory neurons contained within the vagus and glos- sopharyngeal nerves relay information from visceral recep- tors of various modalities to the CNS. The general visceral sensory neurons for the vagus and glossopharyngeal nerves are located in the nodose and petrosal ganglia, respec- tively. The respective general somatic sensory neurons for these two cranial nerves are located in the superior ganglia of the vagus (jugular ganglion) and glossopharyngeal nerves.

The calcium-binding protein, calbindin D-28k, is local- ized to neuronal perikarya in the jugular, petrosal, and

* Corresponding author. Fax: + 1 (301) 295-3220; E-mail: helke@ usuhsb.usuhs.mil

1 The opinions or assertions contained herein are the private ones of the authors and are not to be construed as official or reflecting the views of the DoD or the USUHS. The experiments reported herein were conducted according to the principles set forth in the "Guide for the Care and Use of Laboratory Animals", Institute of Animal Resources, Na- tional Research Council, DHEW Pub. No. (NIH) 74-23.

nodose ganglia [14] where it may act to buffer intracellular calcium levels. Likewise, immunoreactivity a n d / o r mR- NAs for nitric oxide synthase (NOS) and nicotinamide adenosine dinucleotide phosphate (NADPH) diaphorase (a histochemical marker for NOS [6]), are also localized to vagal and glossopharyngeal sensory neurons [1,10,24-27] where the product of NOS, nitric oxide (NO), may act as a novel transmitter agent [3]. Both calbindin D-28k-ir and NADPH-diaphorase activity have been demonstrated in the carotid body and carotid sinus nerve and are thought to be associated with the carotid and vagal chemosensory re- flexes [14,27]. However, little is known about the relation- ship between these substances in the glossopharyngeal and vagal sensory ganglia.

To better understand the potential interactions of these agents in visceral sensory nerve function and the subpopu- lations of visceral sensory neurons containing the agents, we examined the possible coexistence of calbindin D-28k-ir and NADPH-diaphorase activity in the jugular, petrosal, and nodose ganglia. To evaluate the colocalization of calbindin D-28k-ir and NADPH-diaphorase activity in

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326 H. lchikawa, C. J. Helke / Brain Research 735 (1996)325-329

neurotransmitter-identified subpopulations of neurons, we used calcitonin gene-related peptide (CGRP)-ir and tyro- sine hydroxylase (TH)-ir. The presence of CGRP-ir and TH-ir neurons were previously characterized in each gan- glia according to their distributions, peripheral projections, and possible functions [ 11,12,14-16,18].

Six male Sprague-Dawley rats (200-300 g) were deeply anesthetized with chloral hydrate (400 mg/kg , i.p.) and perfused transvascularly (via ascending aorta) with 4% paraformaldehyde in phosphate buffer (pH 7.4). The tissue blocks containing the jugular, petrosal and nodose ganglia were removed and placed in phosphate buffered saline containing 20% sucrose overnight at 4°C. They were frozen, sectioned in a cryostat and mounted on gelatin- coated slides.

Sections of 12-/zm thickness were stained with a single or double immunofluorescence method. For the single immunofluorescence method [5], sections were incubated with mouse monoclonal anti-calbindin D-28k antibody (1:500, Sigma) and fluorescein isothiocyanate conjugated donkey anti-mouse IgG (1:100, Jackson ImmunoResearch Labs.). For the double immunofluorescence method, sec- tions were incubated with a mixture of mouse monoclonal anti-calbindin D-28k antibody and rabbit antiserum for CGRP (1:1000, Cambridge Research Biochemicals) or TH (1:200, Eugene Tech International). Subsequently, sections were incubated with a mixture of fluorescein isothio- cyanate conjugated anti-mouse IgG (1:100) and lissamine rhodamine sulfonyl chloride-conjugated donkey anti-rabbit IgG (1:500, Jackson ImmunoResearch Labs.). The speci- ficities of these antibodies were previously described else- where [11,12,14-16].

Subsequent to photo microscopy of immunofluorescent neurons, the coverslips were removed, and the sections were stained for NADPH-diapborase activity [6]. The sec- tions were incubated in 0.1 M phosphate buffer (pH 7.4) containing 0.1 m g / m l nitroblue tetrazolium (Sigma) and 1.0 m g / m l /3-NADPH (Sigma).

Calbindin D-28k-ir or NADPH-diaphorase positive neu- ronal perikarya were found in the jugular, petrosal, and nodose ganglia. Calbindin D-28k-ir or NADPH-diaphorase positive neurons were of various sizes and contained vari- ous staining intensities. Calbindin D-28k-ir was observed in the neuronal cytoplasm and nucleus, whereas NADPH- diaphorase activity was restricted to the neuronal cyto- plasm.

Calbindin D-28k-ir nerve cells were found in moderate and large numbers, as previously reported [14], in the petrosal and nodose ganglia, respectively. Some calbindin D-28k-ir nerve cells were also observed in the jugular ganglion. Although consistently fewer in number than the calbindin D-28k-ir neurons, NADPH-diaphorase positive nerve cells were numerous in the nodose ganglia, fewer in the jugular ganglion, and rare in the petrosal ganglion. In the jugular and nodose ganglia, there was no apparent rostro-caudal preferences for the localization of either cal-

bindin D-28k-ir or NADPH-diaphorase positive neurons. However, in the petrosal ganglion, calbindin D-28k-ir nerve cells were predominantly found in the caudal portion and NADPH-diaphorase positive neurons in the rostral portion.

Calbindin D-28k-ir and NADPH-diaphorase activity were colocalized in nerve cells in the jugular, petrosal, and nodose ganglia (Fig. 1). A large number ( 7 7 _ 7 neurons/section, mean _+ S.E.M. of n = 5 sections from two ganglia) of calbindin D-28k-ir neurons in the nodose ganglion showed NADPH-diaphorase activity (Fig. 1 and Table 1). Although the percentages of calbindin D-28K-ir neurons colocalizing NADPH-diaphorase activity were similar in the nodose and jugular ganglion, many fewer labeled perikarya were noted in the jugular and petrosal ganglia (Fig. 1A,C and Table 1).

Calbindin D-28k-ir, NADPH-diaphorase activity and CGRP-ir coexisted in nerve cells in the jugular, petrosal, and nodose ganglia (Fig. I A-I) . As described previously [11,12,15], CGRP-ir neurons were mostly localized in the rostral portion of the petrosal and nodose ganglia, and were scattered throughout the jugular ganglion. One-third (rostral nodose ganglion) to nearly two-thirds (petrosal ganglion) of the NADPH-diaphorase positive neurons were also immunoreactive for CGRP (Fig. 1E,F and Table 2). Moreover, significant percentages (25-64%) of the NADPH-diaphorase/CGRP positive neurons additionally colocalized calbindin D-28k-ir (Fig. 1A-I and Table 2). Of the population of calbindin D-28k-ir neurons, only in the jugular ganglion was a significant percentage shown also to be NADPH-diaphorase and CGRP positive (Fig. I A - C and Table 2). In the caudal nodose ganglion, less than 1% of NADPH-diaphorase positive or calbindin D-28k-ir neu- rons were immunoreactive for CGRP, however, most of these colocalized NADPH diaphorase activity and cal- bindin D-28k-ir (Fig. 1G-I).

As described previously [11,12,18], TH-ir nerve cells were clustered in the caudal portion of the petrosal gan- glion and were scattered throughout the nodose ganglion. The jugular ganglion was devoid of TH-ir cells. NADPH- diaphorase positive neurons rarely contained TH-ir in ei- ther the nodose or petrosal ganglion. In the nodose gan- glion, only 5% (34/735 cells counted in five sections of two ganglia) of NADPH-diaphorase positive nerve cells showed TH-ir (Fig. 1K,L).

The present study demonstrated the coexistence of cal- bindin D-28k-ir and NADPH-diaphorase activity in the jugular, petrosal and nodose ganglia of the adult rat. The presence of a calcium-binding protein in significant per- centages of these sensory neurons is in contrast to the rare colocalization of calcium binding proteins, and NADPH- diaphorase activity or NOS-ir in CNS neurons [2,8,13,20]. Whereas studies of this colocalization in other peripheral sensory systems have not been reported, the current data and those obtained from the enteric nervous system [22] suggest that this phenomenon may occur more frequently in the peripheral than CNS.

H. lchikawa, C. J. Helke / Brain Research 735 (1996)325-329 327

In the CNS, both agents are proposed to protect neurons

f rom ischemia , neurotoxic i ty and var ious neurodegenera-

t ive processes [9,19,23,28]. In the nodose gangl ion,

N A D P H - d i a p h o r a s e act ivi ty is increased by axotomy- in-

duced nerve injury [17]. Whi le the funct ions of calbindin

D-28k and N A D P H - d i a p h o r a s e in the vagal and glossopha-

Fig. 1. A-L: photomicrographs of triple staining for calbindin D-28k (A,D,G,J), CGRP (B,E,H), TH (K) and NADPH-diaphorase (C,F,I,L) in the jugular (A-C), rostral nodose (D-F) and caudal nodose ganglia (G-L). Arrows in A-I indicate neurons which colocalize calbindin D-28k-ir, CGRP and NADPH-diaphorase activity. One calbindin D-28k-immunonegative neuron in the caudal nodose ganglion colocalizes TH-ir (K) and NADPH-diaphorase activity (L). The lightly stained neurons in panels H and K show background labeling and not CGRP-ir and TH-ir, respectively. Calibration bar in A indicates 50 /xm. A-L are at the same magnification.

328 H. lchikawa, C. J. Helke / Brain Research 735 (1996) 325-329

Table 1 The percentage of neurons which colocalized NADPH-diaphorase activity and calbindin D-28k-ir in the jugular, petrosal, and nodose ganglia

Jugular Petrosal Nodose

(NADPH-diaphorase + calbindin D-28k) / ( Calbindin D-28k) 23% 2% 27% (35/151) (7/363) (387/1451)

(NADPH-diaphorase + calbindin D-28k) / (NADPH-diaphorase) 41% 33% 51% (35/86) (7/21) (387/758)

The numbers in parentheses indicate the number of positive neurons counted in 14 sections of 4 jugular ganglia, 14 sections of 2 petrosal ganglia, and 5 sections of 2 nodose ganglia.

ryngeal gangl ia are still unclear, the coexis tence o f these

substances suggests a co-opera t ive protect ive action which

may contr ibute to the greater resis tance to nerve injury-in-

duced neuronal death in the peripheral compared to CNS.

Previous studies demonst ra ted that N A D P H - d i a p h o r a s e

posi t ive neurons also contain C G R P in the nodose, dorsal

root, and t r igeminal gangl ia [1]. The present study con-

f i rmed these data in the nodose gangl ion, and extended this

pattern to include the jugu la r and petrosal ganglia. In

addition, we demonst ra ted the coex is tence o f calbindin

D-28k- i r with N A D P H - d i a p h o r a s e act ivi ty in CGRP- i r

neurons. The greatest number o f neurons which colocal-

ized calbindin D-28k-ir , N A D P H - d i a p h o r a s e act ivi ty and

CGRP- i r was found in the jugular ganglion. Because the

jugu la r gangl ion predominant ly contains somatic sensory

neurons o f the pharyngeal and auricular branches o f the

vagus, it is l ikely that jugular gangl ion neurons which

co loca l ized these three substances project to the pharynx

and ear.

Table 2 The percentage of neurons which colocalized NADPH-diaphorase activ- ity, CGRP-ir and/or calbindin D-28k-ir in the jugular, petrosal, and nodose ganglia

Jugular Petrosal Rostral Nodose

(NADPH-diaphorase + CGRP) / (NADPH-diaphorase) 55% 64% 35% (33/60) (9/14) (8/23)

(NADPH-diaphorase + CGRP) / (CGRP) 8% 5% 10% (33/401) (9/18 I) (8/80)

(NADPH-diaphorase + CGRP + calbindin D-28k) / (NADPH-diaphorase + CGRP)

64% 33% 25% (21/33) (3/9) (2/8)

(NADPH-diaphorase + CGRP + calbindin D-28k)/(Calbindin D-28k) 20% 2% 4% (21/106) (3/160) (2/52)

The numbers in each parenthesis indicate the number of positive neurons counted in 8 sections of 2 jugular ganglia, 8 sections of 2 petrosal ganglia, and 4 sections of 2 rostral nodose ganglia.

The nodose gangl ion contained NADPH-d iapho ra se

posi t ive neurons which co loca l ized either calbindin D - 2 8 K

or TH-ir , but not both. Whereas nodose gangl ion neurons

provide visceral afferent innervat ion to the abdominal ,

thoracic and extrathoracic visceral structures, however ,

publ ished reports have descr ibed only the central [4,21,25]

or per ipheral [1,7,10] project ions o f nodose neurons which

contain N A D P H - d i a p h o r a s e or calbindin D-28k. None has

studied their coexis tence in def ined projections. The pet-

rosal gangl ion contains visceral sensory neurons of the

g lossopharyngea l nerve, including the carotid sinus nerve

which innervated the carotid body and carotid sinus [16,18].

The carotid body and carotid sinus nerve contain calbindin

D-28k- i r or N A D P H - d i a p h o r a s e posi t ive nerve fibers

[14,27]. However , the small numbers of neurons in the

petrosal gangl ion found in this study to colocal ize cal-

bindin D-28k- i r and N A D P H - d i a p h o r a s e suggest that cal-

bindin D-28k- i r and N A D P H - d i a p h o r a s e act ivi ty are not

prominent ly co loca l ized in the carotid sinus nerve and

carotid body.

Acknowledgements

This work was supported by N I H Grant R01-NS20991

to C.J.H.

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