immunohistochemical studies on glutamatergic, gabaergic and glycinergic axon varicosities...

Ž .Brain Research 766 1997 72–82

Research report

Immunohistochemical studies on glutamatergic, GABAergic and glycinergicaxon varicosities presynaptic to parasympathetic preganglionic neurons in the

superior salivatory nucleus of the rat

Mariko Kobayashi a,b, Toshimitsu Nemoto a, Hiroshi Nagata a, Akiyoshi Konno a,Tanemichi Chiba b,)

a Department of Otorhinolaryngology, Chiba UniÕersity School of Medicine, 1-8-1 Inohana, Chuoku, Chiba 260, Japanb Department of Anatomy, Chiba UniÕersity School of Medicine, 1-8-1 Inohana, Chuoku, Chiba 260, Japan

Accepted 15 April 1997

Abstract

Ž . Ž .After the superior salivatory nucleus SSN neurons were labeled by administration of cholera toxin B subunit CTB or wheat germŽ .agglutinin conjugated horseradish peroxidase WGA-HRP to the pterygopalatine ganglion, morphological interactions between SSN

neurons and fibers afferent to SSN neurons were examined by light and electron microscopy with double-immunostaining techniques.Antibodies to either the neurotransmitters or its receptor were used to label glutamatergic, GABAergic and glycinergic synapses on theseneurons. By light microscopy, SSN neurons were identified in the ipsilateral ventrolateral part of the rostral medulla oblongata, and rich

Ž .distributions of glutamate- and GABA-immunoreactive ir axon varicosities were observed around SSN neurons. Electron microscopyrevealed that dendrites of SSN neurons received asymmetric synapses from glutamate-ir axon varicosities. Somata as well as dendritesreceived symmetric synapses from GABA-ir varicosities, or showed immunoreactivity for glycine receptors. Quantitative analysis byelectron microscopy showed that glutamate-ir axon varicosities comprised 45.3% of total axon profiles in the SSN region, while GABA-irvaricosities were 20.8% and varicosities presynaptic to glycine receptors were 19.9%. These findings suggest that glutamatergic,GABAergic and glycinergic inputs, originated from a variety of nuclei, directly affect the activity of SSN neurons, and play a role in theregulation of the pterygopalatine ganglion of the rat. q 1997 Elsevier Science B.V.

Keywords: Superior salivatory nucleus; Pterygopalatine ganglion; Glutamate; g-Aminobutyric acid; Glycine receptor; Immunohistochemistry; Choleratoxin B subunit; Wheat germ agglutinin conjugated horseradish peroxidase

1. Introduction

Ž .The superior salivatory nucleus SSN is located in thelower brain stem and consists of the parasympathetic

Ž .preganglionic neurons PPNs projecting to the ptery-Ž .gopalatine ganglion PPG and submandibular ganglion

w x12,36,44,45 . The SSN plays a role in the regulation of theparasympathetic nervous system in the nasal mucosa, oral

Abbreviations: CTB, cholera toxin B subunit; DAB, diaminobenzi-dine; GA, glutaraldehyde; GABA, g-aminobutyric acid; Ir, immunoreac-tive; PB, phosphate buffer; PFA, p-formaldehyde; PPG, pterygopalatineganglion; PPNs, parasympathetic preganglionic neurons; SSN, superiorsalivatory nucleus; TMB, tetramethylbenzidine; WGA-HRP, wheat germagglutinin conjugated horseradish peroxidase.

) Ž .Corresponding author. Fax: q81 43 226-2025; E-mail:[email protected]

mucosa, lacrimal gland and cerebrovascular systems viaw xthe PPG 27,32–34 .

Recently, the afferent innervation of the SSN has beenw xextensively investigated 6,13,14,16,25,42 . Applying a

transganglionic viral transport technique to the PPG of thew xrat, Spencer et al. 42 revealed that various nuclei of the

Ž .central nervous system CNS project to the SSN; they arethe bed nucleus of the stria terminalis, the central nucleusof amygdaloid complex, the paraventricular nucleus of thehypothalamus, lateral hypothalamic area, perifornical area,zona incerta, periaqueductal gray matter, parabrachial nu-cleus, locus coeruleus, spinal trigeminal nucleus, medial

Ž .vestibular nucleus, nucleus of the solitary tract NST ,medullary raphe nuclei, gigantocellular reticular formation

w xpars a and the parapyramidal nucleus. Takeuchi et al. 46demonstrated the direct projection to the SSN from thecentral nucleus of amygdaloid complex by the anterograde

0006-8993r97r$17.00 q 1997 Elsevier Science B.V. All rights reserved.Ž .PII S0006-8993 97 00558-1

( )M. Kobayashi et al.rBrain Research 766 1997 72–82 73

w xtransport of HRP in the cat. Moreover, Hosoya et al. 16showed descending projections from the paraventricularnucleus of the hypothalamus and lateral hypothalamic areato the SSN using Phaseolus Õulgaris leucoagglutininŽ . w xPHA-L and an autoradiographic technique in the rat 14 .In other studies, WGA-HRP injection into the parabrachial

w x 3nucleus of the rat 13 or injections of H-labeled aminow xacids into NST of the cat 25 resulted in the labeling of a

dense network of fibers in the ventrolateral reticular forma-tion of the rostral medulla, which includes SSN. Further-more, direct projections from NST to the reticular forma-tion between NST and the facial nucleus were also demon-strated in the hamster, suggesting that some of them

w xterminate in the SSN 6 . Thus, it has been suggested thatthe activity of SSN neurons is regulated by different typesof inputs from various parts of CNS. Aside from thosestudies regarding projections to the SSN, few immunohis-tochemical studies have specified neurotransmitters con-tained in neurons that provide synapses with SSN neurons.

Ž .We have previously reported that substance P SP ,Ž . Ž .enkephalin ENK , neuropeptide Y NPY , somatostatin

Ž . Ž .SOM , vasoactive intestinal polypeptides VIP , tyrosineŽ . Ž .hydroxylase TH , thyrotropin releasing hormone TRH

Ž .and serotonin 5-HT were found in axon varicositiesw xmaking synaptic contact with SSN neurons 35 . Spencer et

w xal. 42 suggested that the SSN was innervated by some ofthe monoamine cell groups, which were immunoreactive

Ž . Žfor TH A1, A5 and locus coeruleus , serotonin medullaryraphe nuclei, gigantocellular reticular formation pars a

.and parapyramidal nucleus , and histidine decarboxylaseŽ .ventral tuberomammillary nucleus .

However, whether neuronal inputs mediated by majoramino acid transmitters, such as glutamate, GABA orglycine, are present in the SSN has not yet been examined.Therefore, in the present study, we performed immunohis-tochemical analysis of glutamate, GABA and glycine re-ceptors in the rat SSN after labeling SSN neurons by theadministration of CTB or WGA-HRP into the PPG. Theresults showed that considerable numbers of synapses me-diated by those amino acids were distributed in SSNneurons. The functional significance of these amino acidinputs in SSN will be discussed.

2. Materials and methods

2.1. Surgical procedure

Ž .Male adult Sprague-Dawley rats ns25 weighing250–300 g were anaesthetized with sodium pentobarbitalŽ .40 mgrkg, i.p. , and fixed in a headholder of a stereotaxicapparatus. Uni- or bilateral pterygopalatine ganglia were

w xexposed by a zygomatico-lacrimal approach 35,45 . Theganglion is located between the medial surface of themaxillary nerve and the medial orbital wall. To facilitatean easy approach to the whole ganglion, the maxillary

nerve was cut and retracted dorsally. After opening thecapsule, a small cotton-ball presoaked in 0.5 ml of 1%CTB solution or 4% WGA-HRP solution was inserted intothe ganglion. CTB was used for light microscopic studiescombined with glutamate and GABA immunohistochem-istry, and for electron microscopic studies together withglutamate immunohistochemistry. WGA-HRP was usedfor electron microscopic studies combined with GABAand glycine receptor immunohistochemical procedures. Af-ter an interval of 2–7 days, the animals were deeplyreanesthetized and perfused with fixatives through theheart.

Ž .Fixatives used were 2% glutaraldehyde GA in 0.1 Mcacodylate buffer for glutamate immunohistochemistry, 4%

Ž .paraformaldehyde PFA and 2% GA in 0.1 M phosphateŽ .buffer PB for GABA immunohistochemistry and 4%

PFA and 0.2% GA in 0.1 M PB for glycine receptorimmunohistochemistry. After perfusion fixation, brainstems were removed and postfixed at 48C for 2 h in thesame fixatives as used for perfusion.

2.2. Light microscopic immunohistochemistry

Fixed tissues were immersed in 20% sucrose in 0.1 MPB at 48C for 16 h, frozen in liquid nitrogen, and cuttransversely at 20 mm thickness with a cryostat. Thesections were collected in 0.01 M phosphate-buffered salineŽ .PBS , washed three times in PBS, and reduced with 1%sodium borohydride in 0.1 M PB at room temperature for30 min. The sections were washed three times in PBS,preincubated with 1% skim milk and 0.3% Triton X-100 inPBS at room temperature for 60 min, and then incubated at48C for 48–72 h with a mixture of anti-CTB antiserum,and anti-glutamate antibody or anti-GABA antibody. Anti-

ŽCTB was produced in goat List Biological Laboratories.Inc.; diluted 1 : 2000–8000 , and anti-glutamate and anti-

ŽGABA antibodies were produced in rabbit Chemicon;.diluted 1 : 250 and 1 : 200, respectively . The primary anti-

bodies were diluted with the preincubation solution.The sections were rinsed three times in PBS and incu-

Žbated with biotinylated donkey anti-goat IgG GIBCO-.BRL; 1 : 100 in PBS containing 1% skim milk at room

temperature for 2 h. They were then rinsed three times inPBS and incubated in avidin-biotin-peroxidase complexŽ .Vectastain ABC Kit, Vector Laboratories; 1 : 100 in PBSat room temperature for 90 min. After washing thoroughlyin PBS, CTB immunoreactivity was detected by incubation

Ž .in a solution of 0.02% diaminobenzidine DAB in 0.05 MŽ .Tris buffer pH 7.6 containing 0.0006% H O for 8–162 2

min.After washing three times in PBS, the sections were

incubated with peroxidase-labeled anti-rabbit IgG pro-Ž .duced in goat Vector Laboratories; 1 : 400 in PBS at 48C

for 16 h. They were washed and reacted in 0.02% DABsolution containing 0.25% ammonium nickel sulfate hex-ahydrate and 0.0006% H O for 8–14 min.2 2

( )M. Kobayashi et al.rBrain Research 766 1997 72–8274

2.3. Electron microscopic immunohistochemistry

2.3.1. Glutamate immunohistochemistryFor electron microscopic preparations, 9 brain stems

Žwere cut at 40 mm thickness with a Microslicer Dosaka.EM Co. Ltd., Japan . The sections were immersed in 0.1

M PB containing 20% sucrose until sinking to the bottomŽ .of the dish ;1 h . They were then freeze-thawed with

liquid nitrogen, and reduced with 1% sodium borohydrideat room temperature for 30 min. Immunoreaction for gluta-mate and CTB was performed with preincubation solution

w xcontaining 1% skim milk as a blocking agent 35 andŽ . w x0.1% Photo-Flo 600 Kodak 35 as a detergent. Other

steps were performed in the same manner as for lightmicroscopy.

2.3.2. GABA and glycine receptor immunohistochemistryWGA-HRP retrogradely transported from the PPG to

the perikarya of SSN neurons was detected first by theŽ . w xtetramethylbenzidine TMB reactions 29 . Sections were

rinsed with PBS and preincubated with a solution of 0.1%sodium nitroferricyanide and 0.005% TMB in 0.2 M cit-

Ž .rate buffer pH 3.3 for 20 min, followed by the additionof 0.01% H O for 20 min at 48C. After the TMB2 2

reaction, the sections were stabilized in DAB-Co-H O2 2w xsolution 39 for 6–10 min. The sections were carefully

rinsed six times in 0.1 M PB and the immunohistochemicalprocedures for GABA and glycine receptor was carriedout. The sections were preincubated with 1% skim milk inPBS at room temperature for 60 min and then incubated at

Ž .48C for 48–72 h with anti-GABA antibody 1 : 200 orŽanti-glycine receptor antibody mouse, Boehringer

.Mannheim; 1 : 1000 . The primary antibodies were dilutedwith PBS containing 0.1% Photo-Flo 600 and 1% skimmilk. After incubation, the sections were rinsed three timesin PBS and then incubated with biotinylated anti-rabbit

ŽIgG for GABA antibody or anti-mouse IgG goat, Amer-.sham; 1 : 100 for glycine receptor antibody at room tem-

perature for 2 h. The sections were rinsed in PBS andŽincubated in avidin-biotin-peroxidase complex Vectastain

.ABC Kit, Vector Laboratories; 1 : 100 in PBS at roomtemperature for 90 min. After washing thoroughly, theywere reacted in DAB solution, and then the sections werepostfixed with 2% osmium tetroxide in PB for 30 min atroom temperature. The sections were subsequently dehy-drated in increasing concentrations of ethanol and finallyembedded in Spurr’s resin. Ultrathin sections were cut

Ž .with an Ultracut microtome Reichert after trimming andwere stained with aqueous uranyl acetate and lead citrateprior to observation with a JEOL 1200EX electron micro-scope.

2.4. QuantitatiÕe studies

In order to clarify whether or not glutamate-ir andGABA-ir axon varicosities and varicosities presynaptic to

glycine receptors have any tendency to make synapseswith proximal or peripheral dendrites, we measured thediameter of dendrites of SSN neurons postsynaptic toglutamate-ir and GABA-ir axon varicosities, and dendriteswith glycine receptor immunoreactivity. The length of theshortest axis of a dendrite was used as its diameter.

Moreover, in order to determine the density of axonvaricosities immunoreactive for glutamate and GABA, andvaricosities presynaptic to glycine receptors, the number ofaxon varicosities was counted on electron micrographs.Pictures were taken at a direct magnification of =5000and printed at a final magnification of =10 000. Theexamined area was 1656 mm2 for glutamate-ir synapses,1680 mm2 for GABA-ir synapses and 2040 mm2 forsynapses presynaptic to glycine receptors.

In addition, the number of glutamate- and GABA-iraxon varicosities were counted at the light microscopiclevel. For this quantitation, another series of immunohisto-chemical procedures was performed as follows. The brainstems of rats were stained with either anti-phosphate-

Ž .activated glutaminase PAG antibody or anti-GABA anti-body. Procedures for fixation and immunohistochemistrywere largely the same as for light microscopic immunohis-tochemistry as described above. As the fixatives, 4% PFAand 0.2% GA in 0.1 M PB was used for PAG, and 4%PFA and 2% GA in 0.1 M PB was used for GABAimmunohistochemistry. The mouse anti-PAG antibodyŽ .Mab-120; 1 : 200 was provided by Dr Kaneko of Kyoto

w xUniversity 21 . Ten-mm-thick frozen sections were pro-cessed for immunoreactions, and then flatly embedded in

Ž .Spurr’s resin. Semi-thin 1-mm-thick sections were cutfrom the embedded sections. Under a light microscope,neurotransmitter-ir axon varicosities, identified as blue-black dots, were counted. The examined area was 0.12mm2 for PAG-ir and 0.14 mm2 for GABA-ir varicosities.The number of varicosities was standardized by Abercrom-

w xbie’s method 1 .Statistical analysis was performed by the Student’s

t-test, and significant differences are indicated as P-0.05.

3. Results

3.1. Light microscopy

3.1.1. Retrograde labeling of parasympathetic pregan-glionic neurons in the SSN with CTB

After the administration of CTB into the PPG, labeledSSN neurons identified by DAB reaction were found in theipsilateral ventrolateral reticular formation of the rostralmedulla oblongata and caudal pons, from the level of thecaudal part of the superior olive down to the rostral part ofthe facial nucleus. No labeled neurons were observed inthe contralateral side. The great majority of SSN neuronswas found between the facial nucleus and the ventral partof the spinal trigeminal nucleus at the level of the genu of


the facial nerve, and some were seen in the reticularformation just ventral to the facial nucleus. In addition, afew labeled neurons were also encountered within thefacial nucleus. Two to 70 labeled SSN neurons wereobserved in a 20-mm-thick cross section within that region,and the total number of labeled SSN neurons in threeanimals was 712, 688 and 630, respectively. Labeled cellbodies were a multipolar type ranging in size from 9.0=

17.5 mm to 12.6=22.5 mm in the frontal plane, withbranching dendrites extending mainly in the ventrodorsaldirection. These results were mostly the same as those of

w xour previous study 35 .

3.1.2. Glutamate and GABA immunohistochemistryBy light microscopy, amino acid-ir axon varicosities

were identified as bluish-black dots by nickel enhancedDAB reaction products, and were closely associated withdendrites and somata of SSN neurons, which were stained

Ž .brown by DAB reaction products Fig. 1 . Numerousglutamate- and GABA-ir varicosities were seen throughoutthe SSN, with particularly dense distribution in the ventro-lateral part, but no immunoreactive perikarya were ob-

Ž .served. The glutamate-ir axon varicosities arrows, Fig. 1awere rather small in size and were randomly distributed,

while GABA-ir axon varicosities were medium and rela-tively uniform in size. Glutamate-ir varicosities were moreabundant than GABA-ir ones. GABA-ir varicosities weredistributed around both somata and dendrites of SSNneurons, whereas glutamate-ir varicosities were observed

Ž .mainly around dendrites Fig. 1b .

3.2. Electron microscopy

3.2.1. Retrograde labeling of SSN neurons with CTB andWGA-HRP

At the ultrastructural level, CTB-ir neurons were identi-fied by the presence of electron-dense immunoreactionproducts in their cytoplasm. DAB reaction products wereobserved as irregularly assembled dots, and were associ-ated with organella such as microtubules, mitochondria,rough endoplasmic reticulum and Golgi apparatus inperikarya and dendrites. On the other hand, WGA-HRPlabeled neurons were identified by the presence of elec-tron-dense bundles of thin crystalloid reaction products in

Ž . Ž .the cytoplasm Fig. 3c and dendrites Fig. 3a,b,d . Reac-tion products were associated with organelles such asmicrotubules, lysosomes, mitochondria, rough endoplasmicreticulum and Golgi apparatus in perikarya and dendrites.

Ž . Ž .Fig. 1. Photomicrographs showing glutamate-ir a and GABA-ir b axon varicosities, and SSN neurons labeled with CTB applied to the ipsilateral PPG.Ž . Ž .Dense distribution of glutamate-ir varicosities is seen in the SSN a . Glutamate-ir varicosities of small size arrows are often closely associated with CTB

Ž .labeled dendrites. The density of GABA-ir varicosities b, arrows is somewhat less compared with glutamate-ir varicosities. GABA-ir varicosities aremedium and relatively uniform in size. Scale bars20 mm.


3.2.2. Glutamate, GABA and glycine receptor immunohis-tochemistry

Glutamate-ir axon varicosities were easily identified bythe presence of nickel enhanced DAB reaction products

Ž .studding around synaptic vesicles Fig. 2 . Glutamate-irvaricosities contained spherical clear vesicles of approxi-mately 30 nm diameter, and they formed asymmetricsynapses with dendrites that had thick undercoats in theirpostsynaptic membrane.

GABA-ir axon varicosities were identified by granularelectron dense DAB reaction products associated withsynaptic vesicles. They contained pleomorphic synapticvesicles, and formed symmetric synapses with somata as

Ž .well as dendrites of SSN neurons Fig. 3d .Glycine receptor-immunoreactivity was observed as

DAB reaction products in association with the postsynapticŽ .membrane Fig. 4a,b , and was seen in somata as well as

dendrites of SSN neurons. Axon varicosities presynaptic toŽglycine receptors contained flat clear vesicles Fig. 4a,

.inset .Thus, glutamate-ir axon varicosities formed only axo-

dendritic synapses, while GABA-ir varicosities and glycinereceptor immunoreactivities were observed in both axo-dendritic and axo-somatic synapses.

3.3. QuantitatiÕe studies

3.3.1. Thickness of dendrites postsynaptic to glutamate-irand GABA-ir axon Õaricosities, as well as of those im-munoreactiÕe to glycine receptors

The diameter of dendrites postsynaptic to glutamate-iraxon varicosities in the SSN ranged from 0.4 to 3.4 mm,

Ž .with mean and standard deviation mean"S.D. beingŽ .1.16"0.46 mm Fig. 5 . The diameter of dendrites post-

synaptic to GABA-ir axon varicosities ranged from 0.3 toŽ .5.3 mm 1.29"0.53 mm , and that of glycine receptor-ir

Ž .dendrites ranged from 0.2 to 4.3 mm 1.40"0.84 mm .The diameter of dendrites postsynaptic to glutamate-irvaricosities was significantly thinner than that of thosepostsynaptic to GABA and those of glycine receptor-irŽ .P-0.05 .

3.3.2. The density of glutamate-ir and GABA-ir axon Õari-cosities and Õaricosities presynaptic to glycine receptors

The total number of axon varicosities in electron micro-graphs of the SSN of nine rats was counted, and thedensity of varicosities was calculated to be 11.3"7.6r100

2 Ž .mm mean"S.D. . The density of glutamate-ir axonvaricosities was 4.47"3r100 mm2, corresponding to45.3% of the total number of axon varicosities. The den-

Ž . Ž .Fig. 2. Electron micrographs of glutamate-ir axon varicosities in the SSN. Two glutamate-ir axons A1, A2 are seen, one of which A1 forms a synapseŽ . Ž .with a CTB-labeled dendrite D1 . Arrows indicate DAB reaction products of CTB labeling. Inset shows another glutamate-ir axon varicosity A4 forming

Ž .a synapse with a CTB-labeled dendrite D3 . Glutamate-ir axons contain spherical vesicles approximately 30 nm in diameter. Synapses between A1 and D1as well as A4 and D3 are asymmetric type with thick undercoats of postsynaptic membranes. A3; a non-labeled axon varicosity with an asymmetric

Ž .synapse on the CTB-labeled dendrite D1 . D2; a non-labeled dendrite. Scale bars500 nm.


Ž .Fig. 3. Electron micrographs of GABA-ir axon varicosities in the SSN. GABA-ir varicosities A1, A2 and A3 make close contact with a thick dendriteŽ . Ž . Ž .D1, 1.6 mm in diameter , a relatively thin dendrite D2, 1.1 mm in diameter and a somata c . Arrows depict TMB reaction products of WGA-HRP

Ž . Ž .applied to the ipsilateral PPG. N: Nucleus, G: Golgi apparatus, rER: rough endoplasmic reticulum. d is a high power electron micrograph of b . AŽ . Ž .GABA-ir axon varicosity A2 contains pleomorphic clear vesicles and forms a symmetric synapse with a dendrite D2 . Scale bars500 nm.


ŽFig. 4. Electron micrographs of immunocytochemical analysis of glycine receptors. Immunoreactivity for glycine receptors is observed within dendrites a,. Ž .D1, D2 as well as in a somata of SSN neurons b , which are labeled with WGA-HRP applied to the ipsilateral PPG. Arrows indicate TMB reaction

Ž .products of WGA-HRP. rER: rough endoplasmic reticulum. Axon profiles A1 to A4 are presynaptic to glycine receptor-ir portions in dendrites a andŽ . Ž . Ž .somata b . Axon varicosity A5 forms a synapse without glycine receptor immunoreactivity. Inset of a shows flat clear vesicles in an axon varicosity A2

Ž .presynaptic to a glycine receptor-ir dendrite D2 . Scale bars500 nm.


Fig. 5. Populations of axon varicosities glutamate-immunoreactive, GABA-immunoreactive, and those presynaptic to glycine receptors in relation to theŽ .diameter of postsynaptic dendrites of SSN neurons. The diameter of dendrites postsynaptic to glutamate-ir axon varicosities was significantly P-0.05

smaller than that of dendrites postsynaptic to GABA-ir axon varicosities or glycine receptor-ir dendrites. The examined area was 5376 mm2. The meandiameter of dendrites represents the average diameter of all dendrites in the examined area irrespective of amino acid-immunoreactivity.

Table 1Density of glutamate-ir, GABA-ir axon varicosities and axon varicosities presynaptic to glycine receptor-ir dendrites in the rat SSN. The total numberrepresents the varicosities in the area examined for quantitation on electron micrographs

Electron microscopy Light microscopy

Numberr100 Numberrtotal Area examined Numberr Area examined2 2 4 2 2Ž . Ž . Ž .mm synaptic boutons % mm 10 mm mm

Glutamate 4.47"3 45.3 1 656 642"45 0.12GABA 2.6"2.2 20.8 1 680 420"81 0.14Glycine 1.96"0.94 19.9 2 040Total 11.3"7.6

sity of GABA-ir axon varicosities was 2.6"2.2r100 mm2,20.8%. The density of varicosities presynaptic to glycine

2 Ž .receptors was 1.96"0.94r100 mm , 19.9% Table 1 .The density of glutamate-ir axon varicosities was signifi-

Ž .cantly greater than those of the other two types P-0.05 .The number of PAG- and GABA-ir axon varicosities

was counted in six rats by light microscopy, and theirdensities were 642 " 45r10 000 mm2 and 420 "

2 Ž .81r10 000 mm , respectively Table 1 . The density ofglutamate-ir axon varicosities proved to be significantlygreater than that of GABA-ir axon varicosities by light

Ž .microscopic evaluation P-0.05 and supported the dataobtained at the ultrastructural level.

4. Discussion

4.1. Synaptic inputs to parasympathetic preganglionic neu-rons in SSN

SSN is one of the parasympathetic preganglionic nucleiin the CNS and regulates vasodilatation and secretion of

w xglands in the orofacial region via the PPG 31 and sub-w xmandibular ganglia 27 . We previously reported that axon

varicosities making synapses with SSN neurons containedvarious kinds of neurotransmitters, including neuropeptidesand amines such as SP, ENK, NPY, SOM, VIP, TH, TRH

w xand 5-HT 35 . Thus, we speculated that these neurotrans-mitters in variable combinations may play a role in theregulation of the activity of SSN neurons. As an extensionof those studies, we have demonstrated the presence ofglutamatergic, GABAergic, and glycinergic inputs to SSNneurons in the present study. This is the first study thatdemonstrates the presence of amino acid inputs to

Ž .parasympathetic preganglionic neurons PPNs in the ratSSN. Although many studies have shown that glutamater-gic, GABAergic and glycinergic neurons make synapticcontact with sympathetic preganglionic neuronsw x4,8,11,22,24 , the presence of amino acid transmitters inaxons presynaptic to PPNs has been proved in few studies.Batten showed the existence of axon varicosities im-munoreactive to glutamate, GABA and glycine which were

w xpresynaptic to PPNs in the nucleus ambiguus of the cat 5 .


Glutamatergic and glycinergic axonal inputs to neurons ofthe dorsal motor nucleus of vagus in the rat were reportedw x10,40,47,51 .

In the present study, morphological differences in theshape of synaptic vesicles and the type of synapses amongglutamatergic, GABAergic and glycinergic varicositieswere observed. Glutamate-ir axon varicosities containedclear spherical vesicles and formed asymmetric synapses,whereas GABA-ir varicosities contained pleomorphicsynaptic vesicles and formed symmetric synapses, andaxon varicosities presynaptic to glycine receptors con-tained flat clear vesicles. In addition, it was observed thatglutamate-ir varicosities made synapses only with den-drites, while GABA-ir varicosities as well as varicositiespresynaptic to glycine receptors were found to makesynapses with both dendrites and somata. Moreover, it wasdemonstrated that glutamate-ir axon varicosities madesynapses with significantly thinner dendrites than GABA-irvaricosities or varicosities presynaptic to glycine receptors.It is, thus, reasonable to assume that GABAergic andglycinergic axons make synapses mainly with somata andproximal dendrites, and glutamatergic axons form synapsesto more distal parts of dendrites. The morphological fea-tures of amino acid neurons observed in this study weregenerally the same as those observed in other areas of the

w xCNS 2,7,23,38,41,49,50 . A few physiological studies haveshown that glutamate has excitatory, while GABA and

w xglycine have inhibitory effects on PPNs 3,34,48 . There-fore, it seems likely that glutamate is a major excitatoryneurotransmitter, and GABA and glycine are inhibitorytransmitters for PPNs in the rat SSN.

4.2. The density of glutamate-ir and GABA-ir axon Õari-cosities and Õaricosities presynaptic to glycine receptors

We counted the number of glutamate-ir and GABA-iraxon varicosities and varicosities presynaptic to glycine

Ž .receptors on electron micrographs Table 1 , and demon-strated that the density of the former was approximatelytwice that of the latter two. We also showed the number ofglutamatergic varicosities to be more than 1.5 fold ofGABAergic varicosities at the light microscopic level,although the densities of those types were somewhat greaterthan those estimated by electron microscopy.

Additionally, in order to compare the density of axonvaricosities containing amino acids with that of peptidergicaxon varicosities, we also counted the number of SP-irvaricosities by light microscopy. The results showed thatthe density of SP-ir varicosities was 464"97r10 000mm2, slightly larger than that of GABA-ir varicosities and

Ž .much less than that of glutamate-ir varicosities Table 1 .Our previous study showed that the densities of SP- andNPY-ir varicosities were approximately twice that ofENK-ir and SOM-ir varicosities, and far greater than those

w xof VIP-, TRH-, TH- and 5-HT-ir varicosities 35 . Basedon these analyses, it is suggested that glutamatergic,

GABAergic and glycinergic axons comprise a major partof the inputs to PPNs in the rat SSN.

4.3. Origin of neurotransmitter-specific afferents to SSN

w xSpencer et al. 42 demonstrated that a large number ofnuclei of the central autonomic nervous system project toSSN neurons, by injection of pseudorabies virus, atransneuronal retrograde tracer, to the PPG. The nucleiprojecting to the SSN were the bed nucleus of the striaterminalis, central nucleus of amygdaloid complex, par-aventricular nucleus of the hypothalamus, lateral hypotha-lamic area, perifornical area, zona incerta, periaqueductalgray matter, parabrachial nucleus, locus coeruleus, spinaltrigeminal nucleus, medial vestibular nucleus, nucleus ofthe solitary tract, medullary raphe nuclei, gigantocellularreticular formation pars a and parapyramidal nucleus.Other studies employing anterograde tracer techniques alsodemonstrated afferent projections from various regions of

w xthe CNS to the SSN 6,9,13,15,16,18,25,42,46 . The cen-tral nucleus of amygdaloid complex was reported to have a

w x w xdirect projection to the SSN 46 . Hosoya et al. 16 usingPHA-L in the rat showed that paraventricular hypothala-mic nuclei have descending projections to the SSN. Theyalso demonstrated that the lateral hypothalamic area pro-jects directly to the SSN, using 3H-labeled proline as ananterograde tracer and HRP as a retrograde tracer in the ratw x14 . A5 noradrenergic nuclei were also reported to have a

w xconnection with the SSN in the rat 9 . Projections to theŽSSN from parabrachial nucleus especially Kolliker-Fuse¨

. w xnucleus in the rat 13 , and from NST in the cat arew xreported 25 .

w x w xKaneko et al. 19,20 using rat, and Ottersen et al. 37using mouse demonstrated PAG-ir perikarya in the lateralhypothalamic area, paraventricular hypothalamic nucleus,central nucleus of amygdaloid complex, central gray mat-ter in the midbrain and parabrachial nucleus, locuscoeruleus, spinal trigeminal nucleus, NST, medullary raphe,and gigantocellular reticular formation pars a . Glutamate-ircell bodies were also demonstrated in the spinal trigeminal

w xnucleus and medullary raphe of the cat 5 . McDonald etw xal. 28 demonstrated glutamate and aspartate-ir neurons in

projection neurons of the rat amygdala. Therefore, thelateral hypothalamic area, paraventricular hypothalamicnucleus, central nucleus of amygdaloid complex, centralgray matter in the midbrain, parabrachial nucleus, locuscoeruleus, spinal trigeminal nucleus, NST, medullary raphe,and gigantocellular reticular formation pars a are possiblesources of glutamate-ir axon varicosities in synapses withSSN neurons.

Distribution of GABA-ir neurons was demonstrated inthe bed nucleus of the stria terminalis, central nucleus ofamygdaloid complex, central gray matter, perifornical area,locus coeruleus, spinal trigeminal nucleus, NST, medullaryraphe and gigantocellular reticular formation pars a

w x5,26,37,43 . As those nuclei were shown to project to the


SSN by tracer studies, they are possible sources of GABA-ir axons projecting to the SSN. On the other hand, theparagigantocellular nucleus and raphe magnus of reticularformation in the ventral medulla were reported to containcell bodies immunoreactive for both GABA and serotoninw x w x30 . Combined with our previous observations 35 , itseems possible that neurons immunoreactive for bothGABA and serotonin in the ventral medulla project to theSSN. In the locus coeruleus, GABA and TH were reported

w xto coexist within a neuron in the rat 17 , and another studydemonstrated TH-ir neurons in the locus coeruleus project-

w xing to the SSN in the rat 42 . Therefore, such TH- andGABA-ir neurons in the locus coeruleus are also possiblesources of GABAergic inputs to SSN neurons.

As for glycine, central gray matter, spinal trigeminalnucleus and gigantocellular reticular formation pars a

have been reported to contain glycine-ir neurons in the catw x5 . GABA-ir neurons were also reported to reside in those

w xnuclei 37 , and many reports indicated that glycine andGABA have a tendency to coexist in neurons of the CNS

w xin the rat and guinea pig 8,11 . Therefore, it is probablethat sources of glycinergic inputs are similar to those ofGABAergic inputs to SSN neurons.

Acknowledgements

We wish to thank H. Tatsuoka, K. Tanaka, H. Inou, T.Kanda and T. Hanazawa for their valuable advice through-out this study. Thanks are also due to K. Miyama, N. Sakaiand F. Saito for their excellent technical assistance.

References

w x1 M. Abercrombie, Estimation of nuclear population from microtomeŽ .sections, Anat. Rec. 94 1946 239–247.

w x2 P. Andersen, J.C. Eccles, P.E. Voorhoeve, Inhibitory synapses onŽ .somas of Purkinje cells in the cerebellum, Nature 199 1963

655–656.w x3 I. Araki, Inhibitory postsynaptic currents and effects of GABA on

visually identified sacral parasympathetic preganglionic neurons inŽ .neonatal rats, J. Neurophysiol. 72 1994 2903–2910.

w x4 S.B. Backman, J.L. Henry, Effects of GABA and glycine on sympa-thetic preganglionic neurons in the upper thoracic intermediolateral

Ž .nucleus of the cat, Brain Res. 277 1983 365–369.w x5 T.F.C. Batten, Immunolocalization of putative neurotransmitters in-

nervating autonomic regulating neurons of cat ventral medulla, BrainŽ .Res. Bull. 37 1995 487–506.

w x6 M.E. Beckman, M.C. Whitehead, Intramedullary connection of therostral nucleus of the solitary tract in the hamster, Brain Res. 557Ž .1991 265–279.

w x7 D. Bodian, Electron microscopy: two major synaptic types on spinalŽ .motoneurons, Science 151 1966 1093–1094.

w x8 S. Bohlhalter, H. Mohler, J.M. Fritschy, Inhibitory neurotransmis-sion in rat spinal cord: co-localization of glycine- and GABA -re-A

ceptors at GABAergic synaptic contacts demonstrated by tripleŽ .immunofluorescence staining, Brain Res. 642 1994 59–69.

w x9 C.E. Byrum, R. Stornetta, P.G. Guyenet, Afferent and efferent

connections of the A5 noradrenergic cell group in the rat, J. Comp.Ž .Neurol. 261 1987 529–542.

w x10 M.D. Cassell, L. Robert, W.T. Talman, Glycine-containing terminalsŽ .in the rat dorsal vagal complex, Neuroscience 50 1992 907–920.

w x11 T. Chiba, R. Semba, Immuno-electronmicroscopic studies on theg-aminobutyric acid and glycine receptor in the intermediolateralnucleus of the thoracic spinal cord of rats and guinea pigs, J. Auton.

Ž .Nerv. Syst. 36 1991 173–182.w x12 R.J. Contreras, M.M. Gomez, R. Norgren, Central origins of cranial

nerve parasympathetic neurons in the rat, J. Comp. Neurol. 190Ž .1980 373–394.

w x13 H. Herbert, M.M. Moga, C.B. Saper, Connection of parabrachialnucleus with the nucleus of the solitary tract and the medullary

Ž .reticular formation in the rat, J. Comp. Neurol. 293 1990 540–580.w x14 Y. Hosoya, M. Matsushita, Y. Sugiura, A direct hypothalamic

projection to the superior salivatory nucleus neurons in the rat. Astudy using anterograde autoradiographic and retrograde HRP meth-

Ž .ods, Brain Res. 266 1983 329–333.w x15 Y. Hosoya, M. Matsushita, Y. Sugiura, Hypothalamic descending

afferents to cells of origin of the greater petrosal nerve in the rat, asrevealed by a combination of retrograde HRP and anterograde

Ž .autoradiographic techniques, Brain Res. 290 1984 141–145.w x16 Y. Hosoya, Y. Sugiura, R. Ito, K. Kohno, Descending projections

from the hypothalamic paraventricular nucleus to the A5 area,including the superior salivatory nucleus, in the rat, Exp. Brain Res.

Ž .82 1990 513–518.w x17 K. Iijima, M. Sato, N. Kojima, K. Ohtomo, Immunocytochemical in

situ hybridization evidence for the coexistence of GABA and tyro-Ž .sine hydroxylase in the rat locus coeruleus, Anat. Rec. 234 1992

593–604.w x18 A.S.P. Jansen, G.J.T. Horst, T.C. Mettenleiter, A.D. Loewy, CNS

cell groups projecting to the submandibular parasympathetic pregan-glionic neurons in the rat: a retrograde transneuronal viral cell body

Ž .labeling study, Brain Res. 572 1992 253–260.w x19 T. Kaneko, H. Akiyama, I. Nagatsu, N. Mizuno, Immunohistochem-

ical demonstration of glutaminase in catecholaminergic and sero-Ž .toninergic neurons of rat brain, Brain Res. 507 1990 151–154.

w x20 T. Kaneko, K. Itoh, R. Shigemoto, N. Mizuno, Glutaminase-likeimmunoreactivity in the lower brain stem and cerebellum of the

Ž .adult rat, Neuroscience 32 1989 79–98.w x21 T. Kaneko, Y. Urade, Y. Watanabe, N. Mizuno, Production, charac-

terization and immunohistochemical application of monoclonal anti-Ž .bodies to glutaminase purified from rat brain, J. Neurosci. 7 1987

302–309.w x22 W. Laskey, C. Polosa, Characteristics of the sympathetic pregan-

Ž .glionic neuron and its synaptic input, Prog. Neurobiol. 31 198847–84.

w x23 A. Ljungdahl, T. Hokfelt, Autoradiographic uptake patterns of¨w3 x w3 xH GABA and H glycine in central nervous tissues with special

Ž .reference to the cat spinal cord, Brain Res. 62 1973 587–595.w x24 I.J. Llewellyn-Smith, K.D. Phend, J.B. Minson, P.M. Pilowsky, J.P.

Chalmers, Glutamate-immunoreactive synapses on retrogradely-labeled sympathetic preganglionic neurons in rat thoracic spinal

Ž .cord, Brain Res. 581 1992 67–80.w x25 A.D. Loewy, H. Burton, Nuclei of the solitary tract: efferent projec-

tions to the lower brain stem and spinal cord of the cat, J. Comp.Ž .Neurol. 181 1978 421–450.

w x26 B. Maley, B.W. Newton, Immunohistochemistry of gamma-amino-butyric acid in the cat nucleus tractus solitarius, Brain Res. 330Ž .1985 364–368.

w x27 R. Matsuo, T. Yamamoto, Y. Kawamura, Morphological and func-tional evaluation of the superior salivatory nucleus in rabbits, Exp.

Ž .Neurol. 68 1980 147–157.w x28 A.J. McDonald, Glutamate and aspartate immunoreactive neurons of

the rat basolateral amygdala: colocalization of excitatory aminoacids and projections to the limbic circuit, J. Comp. Neurol. 365Ž .1996 367–379.


w x29 M. Mesulam, Tetramethylbenzidine for horseradish peroxidase neu-rohistochemistry: a non-carcinogenic blue reaction product withsuperior sensitivity for visualizing neural afferent and efferent, J.

Ž .Histochem. Cytochem. 26 1978 106–117.w x30 D.E. Millhorn, T. Hokfelt, K. Seroogy, W. Oertel, A.A.J. Verhofs-¨

tad, J.Y. Wu, Extent of colocalization of serotonin and GABA inneurons of the ventral medulla oblongata in rat, Brain Res. 461Ž .1988 169–174.

w x31 H. Motosugi, T. Chiba, A. Konnno, T. Kaneko, Distribution ofneuropeptides in rat pterygopalatine ganglion: light and electronmicroscopic immunohistochemical studies, Arch. Histol. Cytol. 55Ž .1992 513–524.

w x32 T. Murakami, K. Ishizuka, M. Yoshihara, M. Uchiyama, Reflexresponses of single salivatory neurons to stimulation of trigeminal

Ž .sensory branches in the cat, Brain Res. 280 1983 233–237.w x33 T. Murakami, M. Yoshihara, K. Ishizuka, M. Uchiyama, Antidromic

responses and reflex activity of single salivatory neurons in the cat,Ž .Exp. Neurol. 76 1982 218–224.

w x34 M. Nakai, K. Tamiki, J. Ogata, Y. Matsui, M. Maeda, Parasympa-thetic cerebrovasodilator center of the facial nerve, Circ. Res. 72Ž .1993 470–475.

w x35 T. Nemoto, A. Konno, T. Chiba, Synaptic contact of neuropeptide-and amine-containing axons on parasympathetic preganglionic neu-rons in the superior salivatory nucleus of the rat, Brain Res. 685Ž .1995 33–45.

w x36 J.E. Nicholson, C.M. Severin, The superior and inferior salivatoryŽ .nuclei in the rat, Neurosci. Lett. 21 1981 149–154.

w x37 O.P. Ottersen, J. Storm-Mathisen, Neurons containing or accumulat-ing transmitter amino acids, in: A. Bjorklund, T. Hokfelt, M.J.¨ ¨

Ž .Kuhar Eds. , Handbook of Chemical Neuroanatomy, vol. 3, Else-vier, Amsterdam, 1984, pp. 141–246.

w x38 D.L. Price, A. Stocks, J.W. Griffin, A. Young, K. Peck, Glycine-Ž .specific synapses in rat spinal cord, J. Cell Biol. 68 1976 389–395.

w x39 D.B. Rye, C.B. Saper, B.H. Wainer, Stabilization of the tetramethyl-Ž .benzidine TMB reaction product: application for retrograde and

anterograde tracing, and combination with immunohistochemistry, J.Ž .Histochem. Cytochem. 32 1984 1145–1153.

w x40 S. Saha, T.F. Batten, P.N. McWilliam, Glutamate-immunoreactivityin identified vagal afferent terminals of the cat: a study combining

horseradish peroxidase tracing and postembedding electron micro-Ž .scopic immunogold staining, Exp. Physiol. 80 1995 193–202.

w x41 I.R. Schwartz, The differential distribution of label following uptakeof 3H-labeled amino acids in the dorsal cochlear nucleus of the cat,

Ž .Exp. Neurol. 73 1981 601–617.w x42 S.E. Spencer, W.B. Sawyer, H. Wada, K.B. Platt, A.D. Loewy, CNS

projections to the pterygopalatine parasympathetic neurons in therat: a retrograde transneuronal viral cell body labeling study, Brain

Ž .Res. 534 1990 149–169.w x43 J.A. Stamp, K. Semba, Extent of colocalization of serotonin and

Ž .GABA in the neurons of the rat raphe nuclei, Brain Res. 677 199539–49.

w x44 N. Suzuki, J.E. Hardebo, The pathway of parasympathetic nervefibers to cerebral vessels from the otic ganglion in the rat, J. Auton.

Ž .Nerv. Syst. 36 1991 39–46.w x45 N. Suzuki, J.E. Hardebo, G. Skagerberg, C. Owman, Central origins

of preganglionic fibers to the sphenopalatine ganglion in the rat. Afluorescent retrograde tracer study with special reference to itsrelation to central catecholaminergic systems, J. Auton. Nerv. Syst.

Ž .30 1990 101–110.w x46 Takeuchi, Y. Fukui, M. Ichiyama, S. Miyoshi, Y. Nishiura, Direct

amygdaloid projection to the superior salivatory nucleus: a light andŽ .electron microscopic study in the cat, Brain Res. Bull. 27 1991

85–92.w x47 W.T. Talman, S.C. Robertson, M.D. Cassell, Mechanisms of cardio-

vascular responses to glycine injected into the dorsal vagal motorŽŽ .. Ž .nucleus in rat, Hypertension 19 2 Suppl. 1992 II187–II192.

w x48 T.D. Thomson, D.N. Franz, Amino acid and monoamine inhibitionŽ .of sacral parasympathetic preganglionic neurons, in: R. Alan Eds. ,

The Role of Peptides and Amino Acids as Neurotransmitters, LissInc., New York, pp. 181–189.

w x49 K. Uchizono, Characteristics of excitatory and inhibitory synapses inŽ .the central nervous system of the cat, Nature 207 1965 642–643.

w x50 A.N. Van den Pol, T. Gorcs, Glycine and glycine receptor immuno-Ž .reactivity in brain and spinal cord, J. Neurosci. 8 1988 472–492.

w x51 R.J. Washabau, M. Fudge, W.J. Price, F.C. Barone, GABA recep-tors in the dorsal motor nucleus of the vagus influence feline lower

Ž .esophageal sphincter and gastric function, Brain Res. Bull. 38 1995587–594.

immunohistochemical studies on glutamatergic, gabaergic and glycinergic axon varicosities...

Documents