Brain Research, 337 (1985) 127-132 127 Elsevier

BRE 20877

Short Communications

The ultrastructural identification of reticulo-hypoglossal axon terminals anterogradely labeled with horseradish peroxidase

ROSEMARY C. BORKE and MARTIN E. NAU

Department of Anatomy, Uniformed Services University, Bethesda, MD 20814-4799 (U.S.A.)

(Accepted January 28th, 1985)

Key words: nucleus reticularis parvocellularis - - horseradish peroxidase (HRP) - - wheat germ agglutinin-HRP (WGA-HRP) - - anterograde labeling - - axon terminals in hypoglossal nucleus

Injections of horseradish peroxidase (HRP) or wheat-germ agglutinin-horseradish peroxidase (WGA-HRP) into the nucleus reticu- laris parvocellularis (RPc) produced anterograde labeling of axon terminals within the hypoglossal nucleus. Based on morphological parameters of vesicle population, membrane specializations, and postsynaptic articulations, two types of axon terminals derived from neurons in RPc end on hypoglossal neurons. More than half of the terminals contained spherical vesicles (S-type), established asym- metrical membrane specializations and contacted proximal and medium-sized dendrites. The remaining labeled terminals had flat- tened vesicles (F-type), symmetrical membrane densities and apposed medium and small dendrites. The morphological differences expressed in the two types of terminals may reflect physiological and/or pharmacological differences in the action of RPc neurons on motoneurons in the hypoglossal nucleus.

The a r rangement of lingual muscles and reflex

mechanisms of the tongue contr ibute to the highly or-

ganized and control led movements of this muscular

organ. Neural pathways media t ing coordina ted

tongue movements have not been comple te ly identi-

fied, but motoneurons in the hypoglossal nucleus

provide the only source of motor innervat ion to the

muscle mass. Physiological evidence suggests multi-

synaptic pathways control the activity of hypoglossal

motoneurons in mammals o ther than pr imates ; corti- cal regions 20 and cranial nerve ganglia9,11,31, 32 are im-

plicated as sources of first o rde r neurons and second

order neurons reside chiefly in the medulla12,24, 27.

Recently, the anatomical substrates providing direct

hypoglossal input have been de l inea ted in the medul-

la by using re t rograde tracers2, 28. Neurons in the dor-

solateral por t ion of the re t icular format ion, namely

the nucleus reticularis parvocel lular is , consti tute the

pr imary source of the hypoglossal afferents. Physio-

logical data12,20, 24 indicate that this por t ion of the re-

ticular format ion acts as a site for convergence of pe-

r ipheral and cortical fibers and these p remoto r neu-

rons in turn transmit in tegra ted responses to hypo-

glossal motoneurons .

Since RPc assumes a pr imary role in the coordina-

tion of tongue movements , the ways in which RPc

and the hypoglossal nucleus relate structurally to

each other is a basis of interest . HRps,16,17 and

WGA-HRp18 have been used recently as antero-

grade markers to character ize ul trastructural pat-

terns of presynapt ic connectivity; size of axon termi-

nals, vesicle popula t ion , membrane specializations

and postsynaptic art iculations. The object of the cur-

rent investigation was to analyze the distr ibution and

synaptic organizat ion of these ret iculo-hypoglossal

connections and to in terpre t their significance.

Exper iments were carried out in 11 adult , male

rats of the O s b o r n - M e n d e l strain, anesthet ized with

Correspondence: R. C. Borke, Department of Anatomy, Uniformed Services University, 4301 Jones Bridge Road, Bethesda, Mary- land 20814-4799, U.S.A.

0006-8993/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)

128

Fig. 1. A: diffuse, agranular type of HRP label in axon terminal containing spherical vesicles and making synaptic contact at the site of the asymmetrical membrane specialization (arrow) with a medium-sized dendrite (d) in the neuropil of the XIIth nucleus. HRP injec- tion into RPc, 7 days survival, CO-GOD, unstained, × 34,500. B: diffuse, agranular type of HRP label in axon terminal with flattened vesicles. This terminal establishes synaptic contact of the symmetrical type (arrow) with a small dendrite (d) in the neuropil of the

129

an intraperitoneal injection of sodium pentobarbital

(30 mg/kg). For each specimen, the obex served as

the stereotaxic reference point. A glass micropipette (20-30 ktm tip diameter) was introduced in a vertical axis into RPc (lateral to the midlength of the XIIth nucleus) using predetermined stereotaxic coordi-

nates (1.1-1.3 mm lateral, 0.7-0.8 mm anterior, 0.5-0.7 mm depth) relative to the reference point. A

single injection (15-60 nl) of 30% HRP or 1.8% WGA-HRP in 0.1 M Tris buffer was then made

through the micropipette into RPc and the micropi- pette was left in place for 20 min after the injection to minimize the diffusion of the enzyme along the pi- pette track. Animals injected with the tracer were al-

lowed to survive 16-24 h, 2, 3, 5 and 7 days and were reanesthetized and transcardially perfused with Ringer's solution then dilute and concentrated alde- hydes 22 followed by a solution of 0.1 M phosphate buffer. Immediately after perfusion, and each brain removal, the medulla was isolated and mounted in agar. Coronal sections, 50/~m in thickness, were cut on a vibratome. Sample sections were stained with F ink-Heimer and Nauta silver methods 4. The re- maining sections were reacted histochemically for HRP according to the cobalt-glucose oxidase (CO-GOD) 10 or tetramethylbenzidine (TMB) 3,23

methods. Some of the sections reacted for HRP were mounted on gelatinized slides and counterstained with neutral red for light microscopic evaluation of the injection site and localization of areas of antero- grade labeling. Remaining reacted sections were

postfixed in osmium tetroxide, dehydrated and sym- metrical halves of the medulla were embedded in Epon according to previously detailed procedures 3. Epoxy resin blocks were trimmed to include only the hypoglossal nucleus. Ultrathin sections of silver in- terference were cut and left unstained for ultrastruc-

tural evaluation. Light microscopic preparations of HRP reacted

tissue revealed numerous labeled cell bodies and

processes of neurons in RPc. Nerve fibers filled with

reaction product could be traced medially from the injection site into the XIIth nucleus. Occasionally, a few anterogradely labeled fibers traversed the med- ullary raphe below the ventricle and were distributed

to the contralateral nucleus, particularly if the injec- tion extended into the medial aspect of RPc (1.1 mm measurement for lateral stereotaxic coordinate). There was no evidence of direct or indirect damage at the' injection site by the traverse of the pipette or the pressure of the hydraulic injection. Silver staining of vibratome sections adjacent to those reacted for HRP failed to demonstrate anterograde degenera-

tion of terminals in the hypoglossal nucleus or of ax-

ons in RPc. Ultrastructural examination of the hypoglossal nu-

cleus disclosed that the reaction product was found almost exclusively in myelinated axons and axon ter- minals. Both TMB and CO-GOD methods labeled axon terminals containing spherical and flattened vesicles. Quantitative evaluation of 366 CO-GOD- reacted terminals revealed: (1) 56% of labeled termi- nals harbored spherical vesicles (S-type) (Fig. 1A, C, D, E); and (2) 44% of labeled terminals contained flattened vesicles (F-type) (Fig. 1B, F). Asymmet- rical membrane specializations were identified on

78% of the S-type terminals (Fig. 1A, C) and were rarely detected on F-type terminals, which character- istically displayed symmetrical membrane densities (Fig. 1B). The majority of S-type terminals (70%) ended on proximal (Fig. 1E) and medium-sized den- drites (Fig. 1A, C, D). Medium and small dendrites (Fig. 1B, F) served as the primary postsynaptic struc- tures for the F-type terminals (72%). Most of the re- maining S-type terminals contacted small dendrites (16%) whereas F-type terminals apposed proximal dendrites (20%). The postsynaptic structure could not be ascertained for a small percentage (4%) of the total terminals.

The finding of axon terminals making multiple syn-

XIIth nucleus. HRP injection in RPc, 7 days survival, CO-GOD, unstained, × 44,800. C: S-type terminal with diffuse, agranular HRP label, apposes a medium-sized dendrite (d) in the neuropil of the XIIth nucleus. WGA-HRP injection in RPc, 24 h survival, CO- GOD, unstained, x 30,400. D: S-type terminal labeled with a crystal-like aggregate (arrow) when TMB was used as the chromogen. Terminal is in synaptic contact with medium-sized dendrite (d). WGA-HRP injection into RPc, 24 h survival, TMB, unstained, × 34,200. E: membrane-bound, granular type of HRP label (arrows) in S-type terminal. The postsynaptic structure is a proximal den- drite (d) containing ribosomes (r). Other unlabeled terminals of the S-type (s) and the F-type (f) also contact the dendrite. WGA-HRP injection into the RPc, 24 h survival, CO-GOD, unstained, x 27,300. F: membrane-bound, granular type of HRP label (arrow) in F- type terminal contacting a small dendrite (d). WGA-HRP injection into RPc, 42 h survival, CO-GOD, unstained, x 37,000.

130

aptic contacts on proximal and medium-sized den- drites typifies the synaptic relationship of RPc neu- rons ending on other cranial nerve motor nuclei18 and is consistent with data derived from retrograde tracer studies identifying RPc as the source of primary af- ferents of the hypoglossal nucleus2, 28. RPC is thought

to act as the site for convergence and integration of hypoglossal reflexes from spatially separated cortical and peripheral sources. Physiological evidence sug- gests that neurons located in the dorsolateral portion of the reticular formation adjacent to the hypoglossal nucleus may exert either excitatory or inhibitory ef- fects on postsynaptic neurons 12. Although a close re- lationship between vesicle morphology and func- tional synaptic type is not universal, axon terminals containing spherical and flattened vesicles have often been associated respectively with excitatory and inhi- bitory effects on postsynaptic neurons 29. Whether this relationship applies to the current findings is not known. However, the characterization of two mor- phological types of axon terminals may reflect physi- ological and/or pharmacological differences in the actions of RPc neurons on hypoglossal motoneurons.

In TMB-treated sections (Fig. 1D), the reaction product formed crystal-like aggregates that often penetrated membranes and disrupted the morpholo-

gy of the terminals. On the other hand, the ultra- structural integrity of axons and their terminals was preserved with the CO-GOD (Fig. 1A, B, C, E, F) reaction thus confirming other reports TM that this method is preferable for fine structural and semi- quantitative evaluation of synaptic relationships. Af- ter CO-GOD treatment with diaminobenzidine as the chromogen, the distribution of the reaction prod- uct in the axon terminals was characterized by two different patterns. In the majority of terminals, an electron-dense reaction product was applied to the cytomembranes of organelles within the terminals (Fig. 1A-C). This type of labeling has been inter- preted to result primarily from intracytoplasmic dif- fusion of the enzyme into somata and axons damaged by the injection procedure~,5, 6 and has been termed diffuse, agranular labeling. Electron-dense deposits of HRP were detected to a lesser extent within mem- brane-bound vesicles in some terminals (Fig. 1E, F). This kind of enzyme localization represents the in- corporation and transport of the tracer by intact neu- rons in an anterograde direction16.19 and has been

designated membrane-bound, granular label. The types of terminals identified from the antero-

grade tracer study were compared to those charac- terized by use of electron microscopic degeneration techniques. Electrolytic lesions (Fig. 2A) compara- ble in size (<1 mm in diameter) to the HRP injection

sites (Fig. 2B) were placed stereotaxically in RPc of 6 rats. Rats were fixed by transcardiac perfusion as indicated previously. Adjacent vibratome sections of the medulla were processed in a routine manner for plastic embedding or stained with silver stains for an- terograde axonal and terminal degeneration. Marked degenerative changes were detected by light and electron microscopy in RPc and the hypoglossal nucleus. Degenerative changes were recognized mainly in the same types of terminals as those labeled after injection of HRP into RPc: large terminals with spherical (Fig. 2C, D) and flattened (Fig. 2D) vesi- cles made synaptic contact chiefly with large and me- dium and medium and small dendrites, respectively. These changes were apparent in the terminals 1-7 days after lesioning and by 9 days, inclusions con- taining degenerative debris could only be identified in glial cells.

The possibility that some of the labeling of termi- nals in the hypoglossal nucleus after enzyme injec-

tion into RPc resulted from uptake by axons of pass- age was considered. Although neurons of several cell groups in the brainstem project axons that course through RPc 15, only one of these sources, the spinal V nucleus, pars interpolaris and oralis, supplies affer- ent input to the hypoglossal nucleus2. Previous evi- dence indicates that uptake of HRp13, 30 or WGA- HRp21,25,26 by intact axonal shafts does not occur in the CNS. Retrograde cell label, far more common than anterograde label7, was never detected in spinal V neuronal somata suggesting that injured spinal V axons did not account for any of the labeled popula- tion of axon terminals after RPc injections. Further- more, anterograde labeling after injection of HRP into pars interpolaris and oralis portions of the spinal V nucleus in two rats occurred in axon terminals that exhibited morphological features of synaptic organi- zation that could be distinguished from the terminals labeled after the RPc injections. These findings will be reported in a separate communication. The cur- rent findings therefore suggest that the labeled axon terminals originate from neurons in RPc and that the

131

Fig. 2. A: bright-field micrograph of the lesion site (< 1 mm in diameter) in RPc lateral to the hypoglossal nucleus (XII) at a level of the medulla rostral to the obex. 4 days survival, bar = 1 mm. B: dark-field micrograph of an injection site of comparable size and location to the lesion site in A. Neurons of RPc are labeled with enzyme and small particles of labeled substance are situated in the hypoglossal nucleus (XII). No label is seen in the XIIth nerve (n). WGA-HRP, 2 days survival, bar = 1 mm. C: axon terminal of S-type (*) under- going anterograde degeneration in the neuropil of the XIIth nucleus. Electrolytic lesion in RPc, 4 days survival, x 25,g00. D: axon ter- minals of F-type (f) and S-type (s) demonstrating degenerative changes in the neuropil of the XIIth nucleus. Electrolytic lesion in RPc, 4 days survival, x 22,300.

HRP technique can be used to study the ultrastructu-

ral synaptic organization of reticulo-hypoglossal con-

nections.

The author is grateful to Dr. Malcolm Carpenter

for his valuable suggestions and comments. The

skilled typing of Heather Wong is gratefully acknowl-

edged. This work was supported by the Depar tment

of Defense, Uni formed Services University of the

Health Sciences, Depar tment of Defense Grant CO

7019. The opinions or assertions contained herein are

the private ones of the author and are not to be con-

strued 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 Care and Use of Laboratory Animals ' , In-

stitute of Laboratory Animal Resources, National

Research Council, D H E W Pub. No. (NIH) 78-23.

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1 Beattie, M. S., Bresnahan, J. C. and King, J. S., Ultra- structural identification of dorsal root primary afferent ter- minals after retrograde filling with horseradish peroxidase, Brain Research, 153 (1978) 127-134.

2 Borke, R. C., Nau, M. E. and Ringler, R. L. Jr., Brain stem afferents of hypoglossal neurons in the rat, Brain Re- search, 269 (1983) 47-55.

3 Carson, K. A. and Mesulam, M. M., Electron microscopic demonstration of neural connections using horseradish per- oxidase: a comparison of the tetramethylbenzidine proce- dure with other histochemical methods, ]. Histochem. Cy- tochem., 30 (1982) 425-435.

4 Ebbesson, S. O. E., The selective silver impregnation of degenerating axons and their synaptic endings in non-mam- malian species, In W. J. H. Nauta and S. O. E. Ebbesson (Eds.), Contemporary Research Methods in Neuroanato- my, Springer-Verlag, New York, 1970, pp. 132-161.

5 Gobel, S. and Falls, W. M., Anatomical observations of horseradish peroxidase filled terminal primary axonal arbo- rization in layer II of the substantia gelatinosa of Rolando, Brain Research, 175 (1979) 335-340.

6 Gwyn, D. G., Wilkinson, P. H. and Leslie, R. A., The ul- trastructural identification of vagal terminals in the solitary nucleus of the cat after anterograde labeling with horserad- ish peroxidase, Neurosci. Lett., 28 (1982) 139-143.

7 Hedreen, J. C. and McGrath, S., Observations on labeling of neuronal cell bodies, axons and terminals after injection of horseradish peroxidase into rat brain, J. comp. Neurol., 176 (1977) 225-246.

8 Holstege, J. G. and Dekker, J. J., Electron microscopic identification of mammillary body terminals in the rat's AV thalamic nucleus by means of anterograde transport of HRP. A quantitative comparison with the EM degenera- tion and EM autoradiographic techniques, Neurosci. Lett., 11 (1979) 129-135.

9 Hunter, I. W. and Porter, R., Glossopharyngeal influences on hypoglossal motoneurons in the cat, Brain Research, 74 (1974) 161-166.

10 Itoh, K., Konishi, A., Nomura, S., Mizuno, N., Na,kamura, Y. and Sugimoto, T., Application of coupled oxidation re- action to electron microscopic demonstration horseradish peroxidase: cobalt-glucose oxidase method, Brain Re- search, 175 (1979) 341-346.

11 Kaiga, Y., Linguo-hypoglossal reflex elicited by mechani- cal stimulation in rabbits, J. Osaka Odont. Soc., 43 (1980) 449-460.

12 Lowe, A. A., Excitatory and inhibitory inputs to hypoglos- sal motoneurons and adjacent reticular formation neurons in cats, Exp. Neurol., 62 (1978) 30-47.

13 Lynch, G., Gall, C., Mensah, P. and Cotman, C., Horsera- dish peroxidase histochemistry: a new method for tracing efferent projections in the central nervous system, Brain Research, 65 (1974) 373-380.

14 Mawe, G. M., Bresnahan, J. C. and Beattie, M. S., Ultra- structure of HRP-labeled neurons: a comparison of two sensitive techniques, Brain Res. Bull., 10 (1983) 551-558.

15 Mehler, W. R., Observations on the connectivity of the parvicellular-reticular formation with respect to a vomiting center, Brain Behav. Evol., 23 (1983) 63-80.

16 Mizuno, N., Konishi, A., Itoh, K., Iwahori, N. and Naka- mura, Y., Identification of axon terminals of cerebello-oli- vary fibers in the cat: an electron microscopic study using

anterograde horseradish peroxidase method, Neurosci. Lett., 20 (1980) 11-14.

17 Mizuno, N., Nomura, S., Itoh, K., Nakamura, Y. and Ko- nishi, A., Commissural interneurons for masticating moto- neurons: a light and electron microscope study using horse- radish peroxidase tracer technique, Exp. Neurol., 59 (1978) 254-262.

18 Mizuno, N., Yasui, Y., Nomura, S., Itoh, K., Konishi, A., Takada, M. and Kudo, M., A light and electron microscop- ic study of premotor neurons for the trigeminal motor nu- cleus, J. comp. Neurol., 215 (1983) 290-298.

19 Oldfield, B. J., Hou-Yu, A. and Silverman, A. J., Tech- nique for simultaneous ultrastructural demonstration of an- terogradely transported horseradish peroxidase and an im- munocytochemically identified neuropeptide, J. Histo- chem. Cytochem., 31 (1983) 1145-1150.

20 Porter, R., Cortical actions on hypoglossal motoneurons in cats: a proposed role for a common internuncial cell, J. Physiol. (Lond.), 193 (1967) 295-308.

21 Pugh, W. and Kalia, M., Differential uptake of peroxidase (HRP) and peroxidase-lectin (HRP-WGA) conjugate in- jected in the nodose ganglion of the cat, J. Histochem. Cy- tochem., 30 (1982) 887-894.

22 Reese, T. S. and Karnovsky, M. J., Fine structural localiza- tion of a blood brain barrier to exogenous peroxidase, J. Cell Biol., 34 (1967) 207-217.

23 Schonitzer, K. and Hollander, H., Anterograde tracing of horseradish peroxidase (HRP) with the electron micro- scope using the tetramethyl-benzidene reaction, J. Neuro- sci. Meth., 4 (1981) 373-383.

24 Sessle, B. J., Excitatory and inhibitory inputs to single neu- rons in the solitary tract nucleus and adjacent reticular for- mation, Brain Research, 53 (1973) 319-331.

25 Staines, W. A., Kimura, H., Fibiger, H. C. and McGeer, E. G., Peroxidase-labeled lectin as a neuroatomical tracer: evaluation in a CNS pathway, Brain Research, 197 (1980) 485-490.

26 Steindler, D. A., Differences in the labeling of axons of passage by wheat germ agglutinin after uptake by cut pe- ripheral nerve versus injections within the central nervous system, Brain Research, 250 (1982) 159-167.

27 Sumino, R. and Nakamura, Y., Synaptic potential of hypo- glossal motoneurons and a common inhibitory interneuron in the trigemino-hypoglossal reflex, Brain Research, 73 (1974) 439-454.

28 Travers, J. B. and Norgren, R., Afferent projections to the oral motor nuclei in the rat, J. cornp. Neurol., 220 (1983) 280-298.

29 Uchizono, K., Characteristics of excitatory and inhibitory synapses in the central nervous system of the cat, Nature (Lond.), 207 (1965) 642-643.

30 Wakefield, C. and Shonnard, N., Observations of HRP la- beling following injection through a chronically implanted cannula - - a method to avoid diffusion of HRP into injured fibers, Brain Research, 168 (1979) 221-226.

31 Yamamoto, T., Fujiwara, T., Matsuo, R. and Kawamura, Y., Hypoglossal motor nerve activity elicited by taste and thermal stimuli applied to the tongue in rats, Brain Re- search, 238 (1982) 89-104.

32 Yokota, T., Nishikawa, Y. and Ohno, S., A hypoglossal re- flex elicited by mechanical stimulation of the mandibular mucosa in the cat, Jap. J. Physiol., 28 (1978) 659-666.