The anatomical evidence of recurrent axonal collaterals of the thalamus projecting neurons of the rostral pole of the trigeminal sensory nuclear complex in the rat

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Brain Research, 578 (1992) 342-346 © 1992 Elsevier Science Publishers B.V. All rights reserved. 0006-8993/92/$05.00

BRES 25140

The anatomical evidence of recurrent axonal collaterals of the thalamus projecting neurons of the rostral pole of the trigeminal sensory nuclear complex in the rat Pifu Luo Department of Neurobiology, Center of Neuromedicine, Pearl River Hospital, Guangzhou (People's Republic of China) (Accepted 21 January 1992) Key words: Thalamus projecting neuron; Recurrent axon collateral; Horseradish peroxidase; Golgi-like staining; Trigeminal sensory nuclear complex; Rat

Thalamus projecting neurons and their recurrent axonal collaterals were observed in the dorsomedial part of the trigeminal principal sensory nucleus (Vpdm) and the caudolateral part of supratrigeminal nucleus (Vsup CL) after injection of horseradish peroxidase (HRP) into the contralateral ventrobasal complex of the thalamus (VBm) by using the HRP retrogradely tracing-Golgi-like staining method. About 7% (8/120) parent axons of the labeled cells gave rise to recurrent axon collaterals. However, no retrogradely labeled cells were observed in the VBm after injection of HRP into the Vpdm and Vsup CL. In an electron microscopic study, the terminals of recurrent axon collaterals made synapses with the dendrites of the thalamus projecting neurons or non-labeled neurons in the neuropil of the Vpdm and Vsup CL. It is suggested that the recurrent axon collaterals might play a role of negative feedback in transmission of the proprioceptive message from the jaw-closing muscle spindles to the thalamus. It is known that the neurons of the rostral pole of the trigeminal sensory nuclear complex (TSNC), especially, the dorsomedial part of the trigeminal principal sensory nucleus (Vpdm) and the caudolateral part of supratrigeminal nucleus (Vsup CL) project to the medial part of ventrobasal complex of the thalamus (VBm), mesencephalic tectum, cerebellum and motor nuclei of the cranial nerves in the mammalian animals 2'5"6'14-18'2°. Recent studies of the authors demonstrated that these thalamus projecting neurons convey proprioceptive message from the jaw-closing muscle spindles to the contralateral VBm in the rat 7'9. For many years, some investigators have been interested to explore whether the thalamus projecting neurons in the TSNC simultaneously emanate axonal collaterals to the cerebellum, mesencephalic tectum as well as the spinal cord 4"10'18. Unfortunately, their anatomical and electrophysiological studies failed to show the collateralized projections to the cerebellum and spinal cord. A few axon collaterals projected to the contralateral superior colliculus were observed. However, whether these thalamus projecting neurons send recurrent axon collaterals back to their 'own' area of origin is still unknown. The present study was designed to explore the existence of recurrent axon collaterals of the thalamic projecting neurons and their possible roles in

transmission of proprioception of the jaw-closing muscles by using horseradish peroxidase (HRP) retrogradely tracing-Golgi-like staining and light and electron microscopy. The present study is based on data obtained from 18 adult Sprague-Dawley rats. In 10 animals, a single injection of 0.2-0.4/A of 20-30% H R P (Toyobo, dissolved in 2% dimethyl sulfoxide) was placed unilaterally in the VBm via a dorsal approach. Following a postoperative survival time of 48-72 h, the rats were perfused with 0.9% saline (50-100 ml) followed by 300 ml fixative solution (1.0% paraformaldehyde, 1.25% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4) and 0.5% cold sucrose (200 ml). The brains were removed and kept in 30% sucrose in phosphate buffer overnight (4°C), Sections were cut in the transverse plane on a vibratome at a thickness of 50 ktm, reacted with CoCI 2intensified diaminobenzidine ( D A B ) using the method of Adams ~. The sections were divided into two sets. The set for the light microscope study were mounted on chrome alum-gelatin slides, dehydrated in graded alcohols and cleared in xylene. Golgi-like-stained neurons were observed and microphotographs were taken under a microscope (Olympus). The sections for electron microscopy were osmicated

Correspondence: E Luo, Department of Neurobiology, Center of Neuromedicine, Pearl River Hospital, Guangzhou 510282, People's Republic of China.

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Fig. 1. Microphotographs showing individual neurons stained with the Golgi-like method in the Vpdm and Vsup CL after HRP injections of the VBm. A: a multipolar neuron with medium-sized dendritic field; an axon (ax) arising from a primary dendrite. B: a unipolar neuron with a small dendritic field, its dendrite was extented from one pole of the cell body and formed plexiform dendritic trees with numerous beaded varicosities. C: a fusiform neuron with a small dendritic field; its parent axon (ax) was sent from the lateral pole of the cell body; the parent axon (ax) sent a recurrent axonal collateral (ac) at a distance of 50/~m from perikaryon. D: high power microphotograph showing a recurrent axon collateral (ac) arising from its parent axon (ax). Arrows indicate axon (ax) and recurrent axonal collaterals (ac). Bars = 25/~m.

in buffered 1% osmium tetroxide for 1 h, block-stained with saturated uranyl acetate for 8-12 h (4°C), dehyd r a t e d in a graded alcohol series and e m b e d d e d in E p o n 812 on siliconized glass slides. The regions of V p d m and Vsup CL were selected for ultrathin sectioning and observed with a H I T A C H I H-300 electron microscope. In 8 animals, 20-30% H R P was injected into the V p d m and Vsup stereotaxically. A f t e r 48 h, the animals and their brains were treated in the same way as described above and 50 p m thick sections of the contralateral diencephalon containing V B m were cut transversely and reacted with sensitive tetramethyl benzidine (TMB) 12 for light microscopic observation. A large n u m b e r of anterogradely labeled terminals were o b s e r v e d in the contralateral V B m and zona incerta. H o w e v e r , no retrogradely labeled cells were detected either in the V B m or

zona incerta. In the experiments, only 10 cases of H R P injection sites located inside of the V B m were included. Retrogradely labeled neurons were found in the contralateral V p d m and Vsup CL of the trigeminal sensory nuclear complex (TSNC). L a b e l e d neurons stained by the CoC12intensified D A B m e t h o d revealed dense impregnation of the soma, dendrites, dendritic spines and varicosities, axons as well as axonal collaterals (Fig. 1 A - D ) . These were called Golgi-like staining neurons by A r k i n and Miller 3. The details of morphological characteristics and classification of these labeled thalamus projecting neurons have been described elsewhere. Fig. 1 A - D show examples of single V p d m and Vsup C L neurons stained by the Golgi-like method. Fig. 1A illustrates a multipolar neuron with a medium-sized den-

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Fig. 2. Electron microphotographs showing a cell body (S) of a retrogradely labeled neuron stained with the Golgi-like method (A) and terminals (B,C,D)-of its recurrent axonal collaterals. The terminals made synapses with a non-HRP-labeled dendrite (B) and HRP-labeled dendrites (C,D) in the neuropil of the Vpdm and Vsup CL. In C and D, sections showing the same HRP-labeled terminal containing round clear vesicles (A, A1) formed asymmetric synapses with an HRP-labeled dendrite (Den); a non-labeled terminal (A2), containing round clear vesicles, also made synaptic contact with the labeled dendrite; white arrows indicate synaptic sites. Bars, A = 5/~m, B-D = 0.5 ~m.

dritic field, which was located in the border area between the Vpdm and Vsup CL. Some dendrites rapidly taper down to diameters below 0.5/~m and finer, possibly as small as 0.2/am from the perikarya to their distal way. One axon was observed arising from the primary dendrite and coursed dorsolaterally. Fig. 1B illustrates a Vsup CL cell with an ovoid perikaryon and a relatively unipolar, small dendritic field. The parent dendrite gave rise to plexiform dendritic branches with numerous beaded varicosities. The plexiform dendrites directed to the ventromedial direction in the supratrigeminal nucleus. Fig. 1C shows a small dendritic field, fusiform neuron, which was located in the Vpdm. Two primary dendrites arising from the medial pole of the perikaryon. They ramified into 2-3 thin dendritic branches medially

directed, and possessed many beaded varicosities. A smooth thin axon (ax) arose from the cell body, which sent a recurrent axonal collateral at a distance of 50/~m from perikaryon (ac). A b o u t 7% (8/120) of the parent axons of the Vpdm and Vsup CL neurons were identified as sending their recurrent axonal collaterals to the region where their 'own' cell bodies were located. Fig. 1D illustrates a distinct recurrent axonal collateral (ac) arising from the parent axon (ax). In the electron microscope study, the H R P retrogradely labeled neurons had ovoid soma and round nuclei (Fig. 2A). Numerous synaptic contacts between non-labeled terminals and dendritic stem, spines and varicosities were observed w. The most striking feature

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Fig. 3. Electron micrograph of an HRP-labeled terminal (A) from recurrent axon collateral making an asymmetric synapse with a non-HRPlabeled dendrite (Denl); an HRP-labeled dendrite (Den2) was also observed. Black arrowheads indicate synaptic sites. Bar = 0.5 pm.

seen is that some HRP-labeled terminals from the recurrent axonal collaterals stained with the Golgi-like method made synapses with non-labeled dendrites (Figs. 2B and 3) and HRP-labeled dendrites (Fig. 2C,D) in the neuropil of the Vpdm and Vsup CL. In serial ultrathin sections, an HRP-labeled terminal containing clear round vesicles formed an asymmetric synapse with an HRP-labeled dendrite (Fig. 2C,D). Meanwhile, a non-labeled terminal also made synaptic contact with the same HRPlabeled dendrite (Fig. 2D). The neurons of the rostral pole (Vpdm and Vsup CL) of the trigeminal sensory nuclear complex (TSNC) have extensive projections to the thalamus, cerebellum, mesencephalic tectum, spinal cord and contralateral TSNC 46,10,13-18,20. However, previous authors failed to find collateralized projections to the cerebellum as well as the spinal cord. Only a few neurons sent collateralized branches projecting to the superior colliculus contralaterally13,14,16,18. In the previous studies, no report was found about the recurrent axonal collaterals of the thalamus projecting neurons in the Vpdm and Vsup CL, owing to the limitation of the conventional method. The present study,

using the H R P retrogradely tracing-Golgi-like staining technique, identified about 7% of parent axons of the thalamus projecting neurons in the Vpdm and Vsup CL sending their recurrent collaterals to terminate in the areas where their somata were located. Whether these HRP-labeled axon terminals originated from the neurons of the VBm, the present comparative study demonstrated that no retrogradely labeled cells were found in the VBm following H R P injection of the Vpdm and Vsup CL. In the previous studies, no reciprocal connections were mentioned between the Vpdm, Vsup CL and the VBm 12' 15. It is also imperative to clarify the possibility that the axon terminals within the Vsup CL and Vpdm originate from another neuronal population whose axons travel through the VBm. However, no antrogradely labeled terminals were seen to terminate within the Vpdm and Vsup CL in our study, or in previous papers after WGAH R P was injected into the WBm 19. Therefore, it is reasonable to believe that the HRP-labeled terminals in the neuropil of the Vpdm and Vsup CL arose from the recurrent axon collaterals of the thalamus projecting neurons within the nuclei. These terminals made synapses with the dendrites of the thalamus projecting neurons or

346 non-labeled neurons in the neuropil of the Vpdm and Vsup CL. Recent serial studies by the author of this paper indicated that the Vpdm and Vsup CL are relay stations of the proprioceptive messages of jaw-closing muscle spindles transmitting to the thalamus 7'8. F u r t h e r investigations showed that some relay cells of proprioception and their recurrent terminals are G A B A e r g i c 9. In conclusion, the present study suggests that the recur-

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