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Monosynaptic and disynaptic projections from the substantia nigra pars reticulata to the parafascicular thalamic nucleus in the rat
Brain Research 858 Ž2000. 429–435 www.elsevier.comrlocaterbres
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Monosynaptic and disynaptic projections from the substantia nigra pars reticulata to the parafascicular thalamic nucleus in the rat Toshiko Tsumori, Shigefumi Yokota, Hong Lai, Yukihiko Yasui
)
Department of Anatomy (2nd DiÕision), Shimane Medical UniÕersity, Izumo 693-8501, Japan Accepted 10 November 1999
Abstract We examined a direct pathway and an indirect pathway via the reticular thalamic nucleus ŽRT. from the substantia nigra pars reticulata ŽSNr. to the parafascicular thalamic nucleus ŽPF. by using anterograde and retrograde tract tracing methods. After biotinylated dextranamine ŽBDA. injection into the dorsolateral part of the SNr, many labeled fibers and axon terminals were distributed in the ventral part of the RT, as well as in the ventrolateral part of the PF, bilaterally with an ipsilateral dominance. After BDA injection into the ventral part of the RT, a plexus of labeled axons was found bilaterally with an ipsilateral dominance in the ventrolateral part of the PF. After combined injections of BDA into the dorsolateral part of the SNr and cholera toxin B subunit ŽCTb. into the ventrolateral part of the PF on the same side, overlapping distribution of BDA-labeled fibers and CTb-labeled neurons was observed in the ventral part of the RT ipsilateral to the injection sites, where the BDA-labeled axon terminals made symmetrical synaptic contacts with soma and dendrites of the CTb-labeled neurons. q 2000 Elsevier Science B.V. All rights reserved. Keywords: Substantia nigra pars reticulata; Parafascicular thalamic nucleus; Reticular thalamic nucleus; Orofacial movement; Rat
Our previous studies w16,19–22x suggest that the dorsolateral part of the substantia nigra pars reticulata ŽSNr. of the rat takes part in the control mechanism of orofacial motor functions through its descending projections to the parvicellular reticular formation. On the other hand, the SNr has been known to project to the parafascicular nucleus ŽPF. as well as to other thalamic nuclei, such as the ventromedial and mediodorsal nuclei w1,4,14x. Gandia et al. w6x further showed that the SNr sent projection fibers to the ventral part of the reticular thalamic nucleus ŽRT., which has been recently reported to have connections with the midline and intralaminar thalamic nuclei including the PF w9x. The PF gives rise to thalamostriatal projections w2,5x and thalamocortical projections w3,5,11x. Therefore, it is interesting to know how the basal ganglia controls the thalamic flow within the PF with special reference to orofacial motor functions. In the present study, we examined a direct pathway and an indirect pathway relayed by the RT from the dorsolateral part of the SNr to the PF.
)
Corresponding author. Fax: q81-853-20-2105; e-mail: [email protected]
The experiments were carried out on male Wistar rats weighing 230 to 280 g. Operative procedures were performed under general anesthesia following intraperitoneal injection of chloral hydrate Ž350 mgrkg.. An iontophoretic injection of biotinylated dextranamine ŽBDA, Molecular Probes. was made stereotaxically into the SNr in 10 rats or into the RT in 13 rats, using a glass micropipette filled with a 10% BDA solution in 0.01 M phosphate buffer ŽpH 7.3.. The driving current Ž4 to 5 mA, 200-ms duration, 2.0 Hz. was delivered for 10 to 20 min. After 5 to 7 days survival, the rats were reanesthetized and perfused transcardially with 150 ml of saline, followed by 500 ml of a solution composed of 4% paraformaldehyde and 0.1% glutaraldehyde in 0.1 M phosphate buffer ŽpH 7.3., and then with 150 ml of the same buffer containing 10% sucrose. After the perfusion, the brains were removed and placed overnight in a cold solution of 20% sucrose in the same buffer. Subsequently, they were cut serially into frontal sections of 50-mm thickness on a freezing microtome. The sections were washed in phosphate buffered saline ŽPBS., incubated in PBS containing 0.2% Triton X-100 for 3 to 4 h, and then incubated in PBS containing avidine–biotin–peroxidase complex ŽABC, Vector. diluted
0006-8993r00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 6 - 8 9 9 3 Ž 9 9 . 0 2 3 6 8 - 9
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T. Tsumori et al.r Brain Research 858 (2000) 429–435
at 1:100 for 1 h. After washing again in PBS, the sections were incubated in 25 ml of 0.1 M phosphate buffer ŽpH 7.3. containing 10 mg diaminobenzidine and 10 ml of 30% H 2 O2 . Ipsilateral injections of cholera toxin B subunit ŽCTb, List Biol. Labs. into the PF and BDA into the SNr were made stereotaxically by iontophoresis in 18 rats. In each rat, a single injection of BDA into the SNr was made as described above. After BDA injection, a single injection of CTb into the SNr was made through a glass micropipette filled with a 0.5% CTb solution in 0.05 M phosphate buffer ŽpH 6.0., according to Luppi et al. w10x. The driving current Ž4 to 5 mA, 200-ms duration, 2.0 Hz. was delivered for 10 to 15 min. After 5 to 7 days survival, the rats were reanesthetized and perfused transcardially with 150 ml of saline, followed by 500 ml of a solution composed of 4% paraformaldehyde and 0.2% glutaraldehyde in 0.1
M phosphate buffer ŽpH 7.3. and then with 150 ml of 4% paraformaldehyde and 5% glycerin in the same buffer. After perfusion, the brains were removed, postfixed in the same fixative for 5 to 6 h, and immersed overnight in a cold solution of 20% sucrose in the same buffer. Subsequently, serial-frontal sections of the brains were cut at 50 mm on a freezing microtome. The sections were washed in PBS, incubated in PBS containing 0.2% Triton X-100 for 3 to 4 h, and then incubated in PBS containing ABC for 1 h. After washing again in PBS, the sections were incubated in 25 ml of 0.1 M phosphate buffer ŽpH 7.3. containing 10 mg diaminobenzidine, 5 mg nickel ammonium sulfate and 10 ml of 30% H 2 O 2 . BDA-labeled axons were visualized as deep blue to black reaction products. After washing in PBS, the sections were incubated overnight in PBS containing 3% normal rabbit serum, 0.2% Triton X-100 and goat anti-CTb ŽList Biol. Labs, 1:10000.. Subsequently,
Fig. 1. Line drawings showing the distribution of BDA-labeled fibers Žfine lines. and terminals Žsmall dots. in the thalamic region ŽA–E, rostral to caudal. after BDA injection into the dorsolateral part of the SNr Žshaded area in F.. AD, anterodorsal nucleus; AM, anteromedial nucleus; AV, anteroventral nucleus; CG, central gray matter; CL, centrolateral nucleus; CM, central medial nucleus; DLG, dorsal lateral geniculate nucleus; fr, fasciculus retroflexus; G, gelatinosus nucleus; ic, internal capsule; LD, laterodorsal nucleus; LH, lateral habenular nucleus; LP, lateral posterior nucleus; MD, mediodorsal nucleus; ml, medial lemniscus; mt, mammillothalamic tract; PC, paracentral nucleus; PF, parafascicular nucleus; Po, posterior nuclear group; PrC, precommissural nucleus; PV, paraventricular thalamic nucleus; R, red nucleus; Re, reuniens nucleus; RT, reticular thalamic nucleus; SC, superior colliculus; SNr, substantia nigra pars reticulata; SPF, subparafascicular nucleus; VL, ventrolateral nucleus; VM, ventromedial nucleus; VPL, ventral posterolateral nucleus; VPM, ventral posteromedial nucleus; VPMmc, magnocellular division of the VPM; VPMpc, parvicellular division of the VPM; ZI, zona incerta.
T. Tsumori et al.r Brain Research 858 (2000) 429–435
the sections were washed in PBS, incubated in PBS containing biotinylated rabbit anti-goat IgG ŽVector, 1:200. for 3 h, washed again in PBS, and then incubated in PBS containing ABC for 1 h. After washing in PBS, the sections were incubated in 25 ml of 0.1 M phosphate buffer ŽpH 7.3. containing 10 mg diaminobenzidine and 10 ml of 30% H 2 O 2 . CTb-labeled neurons were visualized as brown reaction products. In the sections for electron microscopic observation, BDA was first visualized with diaminobenzidine without nickel ammonium sulfate as mentioned above. After the visualization of BDA, the sections were incubated overnight in PBS containing 1.5% normal rabbit serum, 0.2% Triton X-100 and goat anti-CTb Ž1:10000., washed in PBS and then incubated in PBS containing biotinylated rabbit anti-goat IgG Ž1:200. for 3 h. After several washing with PBS, silver–gold intensification of DAB reaction products of BDA was performed in accordance with the method introduced by Wang and Nakai w18x. Subsequently, the sections were further incubated in the ABC solution and treated with DAB as described above. Specimens, in which there was good overlapping distribution of CTblabeled neurons and BDA-labeled axon terminals, were cut out from the PF region and collected in 0.1 M phosphate buffer ŽpH 7.3.. They were postfixed with a solution of 2% osmium tetroxide in the same buffer for 30 min at room temperature. After washing in distilled water, the specimens were stained en bloc with 0.5% uranyl acetate in
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70% ethanol for 1 h, dehydrated in a graded series of ethanol, cleared in propylene oxide and then embedded flat in Epon. Subsequently, serial ultrathin sections were cut on an ultramicrotome, collected on collodion-coated grids and stained with lead nitrate. Finally, the sections were examined under an electron microscope ŽJEOL, JEM 1200EX. and photographed. The minor diameters of CTb-labeled dendrites postsynaptic to BDA-labeled terminals with synaptic membrane specializations were measured on the photomicrographs by using an image analysing system ŽNikon, COSMOZONE-1S.. After BDA injection into the dorsolateral part of the SNr ŽFig. 1F., labeled fibers and axon terminals were distributed bilaterally with a clear-cut ipsilateral dominance in the thalamic region ŽFig. 1A–E.. A dense plexus of labeled fibers and terminals was observed in the ventral part of the RT slightly caudal to its rostral pole, mainly along the lateral margin ŽFig. 1A. although labeling was less dense in the rostralmost part of the RT. At the levels of the rostral two-thirds of the entopeduncular nucleus, dense labeling in the ventral part of the RT was found along both the medial and the lateral margins ŽFig. 1B.. Light labeling was seen around the border between the ventromedial and the ventrolateral nuclei. At more caudal levels, labeling in the RT was sparse although the dorsal part of the ventromedial nucleus contained a very dense plexus of labeled axons ŽFig. 1C.. Moderate to dense labeling was found in the lateralmost part of the central
Fig. 2. Line drawings showing the site of BDA injection into the ventral part of the RT Žshaded area indicated by a curbed arrow in A. and resulting anterograde labeling in the PF ŽB–D, rostral to caudal.. BDA-labeled fibers and terminals were represented by fine lines and small dots, respectively. APTD, dorsal part of the anterior pretectal nucleus; CPu, caudate putamen; f, fornix; GP, globus pallidus; opt, optic tract. Other abbreviations are as in Fig. 1.
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medial nucleus and in the medialmost parts of the centrolateral and ventrolateral nuclei, whereas labeling was sparse in the paralaminar portion of the mediodorsal nucleus. In the caudal thalamic region, many labeled fibers and terminals were distributed in the ventrolateral part of the PF with a few labeled axons in its ventromedial part ŽFig. 1D, E.. In the second set of experiments, BDA was injected into the ventral part of the RT ŽFig. 2A., where labeled axon terminals had been found after BDA injection into the dorsolateral part of the SNr. Reticular projections were predominantly uncrossed. Dense labeling was found in the ventromedial part of the ventrolateral nucleus, the dorsal part of the ventromedial nucleus and the adjacent parts of the intralaminar nuclei. Light labeling was seen in the ventralmost part of the mediodorsal nucleus. Labeled fibers
with bouton-like varicosities were also distributed in the PF: a moderate to dense plexus of labeled axons was observed not only in the rostralmost part of the PF ŽFig. 2B. but also more caudally in the ventrolateral part of the PF ŽFig. 2C, D.. The plexus extended into the ventromedial part of the posterior thalamic nuclear group, giving rise to light labeling. In the third set of experiments, CTb injection into the ventrolateral part of the PF ŽFigs. 3A and 4A. and BDA injection into the dorsolateral part of the SNr ŽFigs. 3B and 4B. were made on the same side. The distribution pattern of BDA-labeled fibers and terminals in the RT was similar to that in the rats of the first set of experiments. CTb-labeled cell bodies were distributed predominantly in the ventral part of the RT, bilaterally with an ipsilateral dominance. Labeled cell bodies were also found in an area just ventro-
Fig. 3. Line drawings illustrating the sites of CTb injection into the ventrolateral part of the PF Žshaded area in A. and BDA injection into the dorsolateral part of the SNr Žshaded area in B., and resulting distributions of BDA-labeled fibers Žfine lines. and terminals Žsmall dots., and CTb-labeled neurons Žlarge dots. in the RT ŽC–F, rostral to caudal.. Each area enclosed by the small rectangle in the thalamic planes Žc–f. is enlarged in C–F, respectively. MG, medial geniculate nucleus; STh, subthalamic nucleus. Other abbreviations are as in Figs. 1 and 2.
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Fig. 4. Photomicrographs representing the sites of CTb injection into the ventrolateral part of the PF ŽA. and BDA injection into the dorsolateral part of the SNr ŽB., and resulting overlapping distribution of CTb-labeled neurons and BDA-labeled fibers in the ventral part of the RT ŽC,D.. Note bouton-like varicosities labeled with BDA Žarrow heads. tightly surrounding the somata ŽC. and distal dendrites ŽD. of CTb-labeled neurons. Abbreviations are as in Figs. 1 and 3. Bar s1 mm in A,B; 20 mm in C,D.
medial to the rostral portion of the RT, in the lateralmost part of the zona incerta, and within the internal capsule. In the rostral portion of the RT, some labeled cell bodies were seen mainly along the lateral margin ŽFig. 3C.. At the level of the entopedunclular nucleus, the ventral part of the RT contained many labeled cell bodies, most of which were located in the medialmost part or along the lateral margin ŽFig. 3D–F.. More caudally, labeled cells in the RT gradually disappeared. Most of these CTb-labeled neurons were embedded in a plexus of BDA-labeled axon terminals within the ventral part of the RT, where bouton-
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like varicosities labeled with BDA were observed in apposition with cell bodies or proximal dendrites ŽFig. 4C. as well as with distal dendrites ŽFig. 4D. of CTb-labeled neurons. At the electron microscopic level, CTb-labeled neurons were identified by the presence of the electrondense DAB reaction products, which were dispersed not only throughout somatic and large dendritic profiles but also within small dendritic profiles. On the other hand, BDA-labeled fibers and terminal boutons were packed with dense grains of various sizes filling up the cytoplasm except mitochondria. As far as identified, BDA-labeled terminals made symmetrical synaptic contacts with dendrites ŽFig. 5A, B. as well as with soma ŽFig. 5C. of the RT neurons, most of which were labeled with CTb. Out of 48 BDA-labeled terminals forming synaptic contacts with CTb-labeled neurons, which were identified on the photomicrographs, 19 and 29 terminals were in contact with soma and dendrites, respectively. The minor diameter of CTb-labeled dendrites postsynaptic to BDA-labeled terminal boutons ranged from 0.5 to 2.2 mm with a mean of 1.22 mm " 0.37 S.D. The present study indicates that the dorsolateral part of the SNr projects not only monosynaptically but also disynaptically via the ventral part of the RT to the ventrolateral part of the PF. As mentioned in the introduction, the dorsolateral part of the SNr has been considered to contribute to the control mechanism of orofacial movements. Therefore, the nigrothalamic pathways observed here may also be implicated in this mechanism. The nigrothalamic projections from the SNr to the PF have been revealed by using tracer methods w1,4,14x. The present results further showed that the dorsolateral part of the SNr projected to the ventrolateral part of the PF. According to our preliminary study w15x, this part of the PF sends projection fibers to the ventrolateral part of the striatum as well as to the orofacial motor cortex. In addition, the ventrolateral part of the striatum has been known to receive projection fibers from the orofacial motor cortex w23x and to send projection fibers to the dorsolateral part of the SNr w17x. Altogether, these data suggest that within the orofacial motor circuits, the ventrolateral part of the PF is a key station of the thalamic flow to the cerebral cortex and striatum and is under the influence of output neurons of the basal ganglia. An anterograde WGA-HRP study w6x indicated that the SNr sent projection fibers to the ventral part of the rostral two-thirds of the RT. Recently, Kolmac and Mitrofanis w9x reported that neurons in this RT area were retrogradely labeled with biotinylated dextran injected into the PF as well as into other intralaminar nuclei. In the present study, we confirmed these observations by showing the overlapping distribution of the nigral fibers originating from the dorsolateral part of the SNr and the RT neurons projecting to the ventrolateral part of the PF in the ventral part of the RT, and further demonstrated that synaptic connections were made between these nigral fibers and RT neurons.
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Fig. 5. Electron micrographs showing the synaptic connections between the CTb-labeled neurons and the BDA-labeled axon terminals in the ventral part of the RT after combined injections of CTb into the PF and BDA into the SNr. ŽA. Two BDA-labeled terminals Žt1, t2. forming symmetrical synaptic contacts with a dendritic profile Žd1. of the CTb-labeled neuron. A non-labeled dendritic profile Žd2. is also seen. ŽB. Another example of a symmetrical synaptic contact between a BDA-labeled terminal Žt. and a dendritic profile Žd. of the CTb-labeled neuron. ŽC. A BDA-labeled terminal Žt. making a symmetrical synaptic contact with a somatic profile Žs. of the CTb-labeled neuron. Arrowheads indicate symmetrical synaptic specialization. The DAB reaction products are presented by arrows. Bar s 0.5 mm.
The g-aminobutyric acid ŽGABA. neurons in the SNr are well known to send their axons to the thalamus Žsee Ref. w7x for review., and therefore the direct SNr-PF pathway may provide an inhibitory input to PF neurons. Since neurons in the RT are also GABAergic w8,12,13x, the indirect SNr-PF pathway relayed by the RT may exert disinhibitory influence on PF neurons through the inhibitory influence of the SNr on inhibitory RT neurons projecting to the PF. If the SNr exerts its influence simultaneously on PF neurons and PF-projecting RT neurons, PF neurons receiving SNr fibers might be different from those receiving axons of SNr-recipient RT neurons because it seems unlikely that single PF neurons are under the inhibitory and disinhibitory influences of the SNr at the same time. Thus, the direct SNr-PF and indirect SNr-RT-PF pathways, revealed by the present study, may regulate the
thalamic output stream to the cerebral cortex andror striatum by inhibition and disinhibition of PF neurons, respectively, in the control of orofacial movements.
Acknowledgements The authors are grateful for the support of Drs. Ryuzo Fujimoto, Kazuya Fujita, Yasuichi Munenaga, Takashi Sakurai, Yayoi Sakurai, Hiromi Taguchi and Katsumi Tanaka. This study was supported in part by Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan ŽNos. 09832009, 11680740., and by a grant from the Shimane Medical University Education and Research Foundation.
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w13x P.T. Ohara, A.R. Liebermann, The thalamic reticular nucleus of the adult rat: experimental anatomical studies, J. Neurocytol. 14 Ž1985. 365–411. w14x S.T. Sakai, I. Grofova, K. Bruce, Nigrothalamic projections and nigrothalamocortical pathway to the medial agranular cortex in the rat: single- and double-labeling light and electron microscopic studies, J. Comp. Neurol. 391 Ž1998. 506–525. w15x T. Tsumori, K. Ono, S. Yokota, T. Kishi, Y. Yasui, Projections from the substantia nigra pars reticulata to the cortex and the striatum relayed by the parafascicular thalamic nucleus in the rat, Neurosci. Res. Suppl. 22 Ž1998. S172. w16x T. Tsumori, Y. Yasui, Organization of the nigro-tecto-bulbar pathway to the parvicellular reticular formation: a light- and electron-microscopic study in the rat, Exp. Brain Res. 116 Ž1997. 341–350. w17x M. von Krosigk, Y. Smith, J.P. Bolam, A.D. Smith, Synaptic organization of GABAergic inputs from the striatum and the globus pallidus onto neurons in the substantia nigra and retrorubral field which project to the medullary reticular formation, Neuroscience 50 Ž1992. 531–549. w18x Q.-P. Wang, Y. Nakai, Double preembedding immunostaining for the study by electron microscopy of synaptic relationships between immunocytochemically identified neurons, Neurosci. Prot. 96-050-05 Ž1996. 1–13. w19x Y. Yasui, Descending pathways from the basal ganglia to the orofacial premotor neuron pools, in: T. Morimoto, T. Matsuya, K. Takada ŽEds.., Brain and Oral Functions, Elsevier, Amsterdam, 1995, pp. 77–85. w20x Y. Yasui, K. Nakano, Y. Nakagawa, T. Kayahara, T. Shiroyama, N. Mizuno, Non-dopaminergic neurons in the substantia nigra project to the reticular formation around the trigeminal motor nucleus in the rat, Brain Res. 585 Ž1992. 361–366. w21x Y. Yasui, T. Tsumori, A. Ando, T. Domoto, T. Kayahara, K. Nakano, Descending projections from the superior colliculus to the reticular formation around the motor trigeminal nucleus and the parvicellular reticular formation of the medulla oblongata in the rat, Brain Res. 656 Ž1994. 420–426. w22x Y. Yasui, T. Tsumori, K. Ono, T. Kishi, Nigral axon terminals are in contact with parvicellular reticular neurons which project to the motor trigeminal nucleus in the rat, Brain Res. 775 Ž1997. 219–224. w23x G. Zhang, K. Sasamoto, Projections of two separate cortical areas for rhythmical jaw movements in the rat, Brain Res. Bull. 24 Ž1990. 221–230.
Short communication
Monosynaptic and disynaptic projections from the substantia nigra pars reticulata to the parafascicular thalamic nucleus in the rat Toshiko Tsumori, Shigefumi Yokota, Hong Lai, Yukihiko Yasui
)
Department of Anatomy (2nd DiÕision), Shimane Medical UniÕersity, Izumo 693-8501, Japan Accepted 10 November 1999
Abstract We examined a direct pathway and an indirect pathway via the reticular thalamic nucleus ŽRT. from the substantia nigra pars reticulata ŽSNr. to the parafascicular thalamic nucleus ŽPF. by using anterograde and retrograde tract tracing methods. After biotinylated dextranamine ŽBDA. injection into the dorsolateral part of the SNr, many labeled fibers and axon terminals were distributed in the ventral part of the RT, as well as in the ventrolateral part of the PF, bilaterally with an ipsilateral dominance. After BDA injection into the ventral part of the RT, a plexus of labeled axons was found bilaterally with an ipsilateral dominance in the ventrolateral part of the PF. After combined injections of BDA into the dorsolateral part of the SNr and cholera toxin B subunit ŽCTb. into the ventrolateral part of the PF on the same side, overlapping distribution of BDA-labeled fibers and CTb-labeled neurons was observed in the ventral part of the RT ipsilateral to the injection sites, where the BDA-labeled axon terminals made symmetrical synaptic contacts with soma and dendrites of the CTb-labeled neurons. q 2000 Elsevier Science B.V. All rights reserved. Keywords: Substantia nigra pars reticulata; Parafascicular thalamic nucleus; Reticular thalamic nucleus; Orofacial movement; Rat
Our previous studies w16,19–22x suggest that the dorsolateral part of the substantia nigra pars reticulata ŽSNr. of the rat takes part in the control mechanism of orofacial motor functions through its descending projections to the parvicellular reticular formation. On the other hand, the SNr has been known to project to the parafascicular nucleus ŽPF. as well as to other thalamic nuclei, such as the ventromedial and mediodorsal nuclei w1,4,14x. Gandia et al. w6x further showed that the SNr sent projection fibers to the ventral part of the reticular thalamic nucleus ŽRT., which has been recently reported to have connections with the midline and intralaminar thalamic nuclei including the PF w9x. The PF gives rise to thalamostriatal projections w2,5x and thalamocortical projections w3,5,11x. Therefore, it is interesting to know how the basal ganglia controls the thalamic flow within the PF with special reference to orofacial motor functions. In the present study, we examined a direct pathway and an indirect pathway relayed by the RT from the dorsolateral part of the SNr to the PF.
)
Corresponding author. Fax: q81-853-20-2105; e-mail: [email protected]
The experiments were carried out on male Wistar rats weighing 230 to 280 g. Operative procedures were performed under general anesthesia following intraperitoneal injection of chloral hydrate Ž350 mgrkg.. An iontophoretic injection of biotinylated dextranamine ŽBDA, Molecular Probes. was made stereotaxically into the SNr in 10 rats or into the RT in 13 rats, using a glass micropipette filled with a 10% BDA solution in 0.01 M phosphate buffer ŽpH 7.3.. The driving current Ž4 to 5 mA, 200-ms duration, 2.0 Hz. was delivered for 10 to 20 min. After 5 to 7 days survival, the rats were reanesthetized and perfused transcardially with 150 ml of saline, followed by 500 ml of a solution composed of 4% paraformaldehyde and 0.1% glutaraldehyde in 0.1 M phosphate buffer ŽpH 7.3., and then with 150 ml of the same buffer containing 10% sucrose. After the perfusion, the brains were removed and placed overnight in a cold solution of 20% sucrose in the same buffer. Subsequently, they were cut serially into frontal sections of 50-mm thickness on a freezing microtome. The sections were washed in phosphate buffered saline ŽPBS., incubated in PBS containing 0.2% Triton X-100 for 3 to 4 h, and then incubated in PBS containing avidine–biotin–peroxidase complex ŽABC, Vector. diluted
0006-8993r00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 6 - 8 9 9 3 Ž 9 9 . 0 2 3 6 8 - 9
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at 1:100 for 1 h. After washing again in PBS, the sections were incubated in 25 ml of 0.1 M phosphate buffer ŽpH 7.3. containing 10 mg diaminobenzidine and 10 ml of 30% H 2 O2 . Ipsilateral injections of cholera toxin B subunit ŽCTb, List Biol. Labs. into the PF and BDA into the SNr were made stereotaxically by iontophoresis in 18 rats. In each rat, a single injection of BDA into the SNr was made as described above. After BDA injection, a single injection of CTb into the SNr was made through a glass micropipette filled with a 0.5% CTb solution in 0.05 M phosphate buffer ŽpH 6.0., according to Luppi et al. w10x. The driving current Ž4 to 5 mA, 200-ms duration, 2.0 Hz. was delivered for 10 to 15 min. After 5 to 7 days survival, the rats were reanesthetized and perfused transcardially with 150 ml of saline, followed by 500 ml of a solution composed of 4% paraformaldehyde and 0.2% glutaraldehyde in 0.1
M phosphate buffer ŽpH 7.3. and then with 150 ml of 4% paraformaldehyde and 5% glycerin in the same buffer. After perfusion, the brains were removed, postfixed in the same fixative for 5 to 6 h, and immersed overnight in a cold solution of 20% sucrose in the same buffer. Subsequently, serial-frontal sections of the brains were cut at 50 mm on a freezing microtome. The sections were washed in PBS, incubated in PBS containing 0.2% Triton X-100 for 3 to 4 h, and then incubated in PBS containing ABC for 1 h. After washing again in PBS, the sections were incubated in 25 ml of 0.1 M phosphate buffer ŽpH 7.3. containing 10 mg diaminobenzidine, 5 mg nickel ammonium sulfate and 10 ml of 30% H 2 O 2 . BDA-labeled axons were visualized as deep blue to black reaction products. After washing in PBS, the sections were incubated overnight in PBS containing 3% normal rabbit serum, 0.2% Triton X-100 and goat anti-CTb ŽList Biol. Labs, 1:10000.. Subsequently,
Fig. 1. Line drawings showing the distribution of BDA-labeled fibers Žfine lines. and terminals Žsmall dots. in the thalamic region ŽA–E, rostral to caudal. after BDA injection into the dorsolateral part of the SNr Žshaded area in F.. AD, anterodorsal nucleus; AM, anteromedial nucleus; AV, anteroventral nucleus; CG, central gray matter; CL, centrolateral nucleus; CM, central medial nucleus; DLG, dorsal lateral geniculate nucleus; fr, fasciculus retroflexus; G, gelatinosus nucleus; ic, internal capsule; LD, laterodorsal nucleus; LH, lateral habenular nucleus; LP, lateral posterior nucleus; MD, mediodorsal nucleus; ml, medial lemniscus; mt, mammillothalamic tract; PC, paracentral nucleus; PF, parafascicular nucleus; Po, posterior nuclear group; PrC, precommissural nucleus; PV, paraventricular thalamic nucleus; R, red nucleus; Re, reuniens nucleus; RT, reticular thalamic nucleus; SC, superior colliculus; SNr, substantia nigra pars reticulata; SPF, subparafascicular nucleus; VL, ventrolateral nucleus; VM, ventromedial nucleus; VPL, ventral posterolateral nucleus; VPM, ventral posteromedial nucleus; VPMmc, magnocellular division of the VPM; VPMpc, parvicellular division of the VPM; ZI, zona incerta.
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the sections were washed in PBS, incubated in PBS containing biotinylated rabbit anti-goat IgG ŽVector, 1:200. for 3 h, washed again in PBS, and then incubated in PBS containing ABC for 1 h. After washing in PBS, the sections were incubated in 25 ml of 0.1 M phosphate buffer ŽpH 7.3. containing 10 mg diaminobenzidine and 10 ml of 30% H 2 O 2 . CTb-labeled neurons were visualized as brown reaction products. In the sections for electron microscopic observation, BDA was first visualized with diaminobenzidine without nickel ammonium sulfate as mentioned above. After the visualization of BDA, the sections were incubated overnight in PBS containing 1.5% normal rabbit serum, 0.2% Triton X-100 and goat anti-CTb Ž1:10000., washed in PBS and then incubated in PBS containing biotinylated rabbit anti-goat IgG Ž1:200. for 3 h. After several washing with PBS, silver–gold intensification of DAB reaction products of BDA was performed in accordance with the method introduced by Wang and Nakai w18x. Subsequently, the sections were further incubated in the ABC solution and treated with DAB as described above. Specimens, in which there was good overlapping distribution of CTblabeled neurons and BDA-labeled axon terminals, were cut out from the PF region and collected in 0.1 M phosphate buffer ŽpH 7.3.. They were postfixed with a solution of 2% osmium tetroxide in the same buffer for 30 min at room temperature. After washing in distilled water, the specimens were stained en bloc with 0.5% uranyl acetate in
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70% ethanol for 1 h, dehydrated in a graded series of ethanol, cleared in propylene oxide and then embedded flat in Epon. Subsequently, serial ultrathin sections were cut on an ultramicrotome, collected on collodion-coated grids and stained with lead nitrate. Finally, the sections were examined under an electron microscope ŽJEOL, JEM 1200EX. and photographed. The minor diameters of CTb-labeled dendrites postsynaptic to BDA-labeled terminals with synaptic membrane specializations were measured on the photomicrographs by using an image analysing system ŽNikon, COSMOZONE-1S.. After BDA injection into the dorsolateral part of the SNr ŽFig. 1F., labeled fibers and axon terminals were distributed bilaterally with a clear-cut ipsilateral dominance in the thalamic region ŽFig. 1A–E.. A dense plexus of labeled fibers and terminals was observed in the ventral part of the RT slightly caudal to its rostral pole, mainly along the lateral margin ŽFig. 1A. although labeling was less dense in the rostralmost part of the RT. At the levels of the rostral two-thirds of the entopeduncular nucleus, dense labeling in the ventral part of the RT was found along both the medial and the lateral margins ŽFig. 1B.. Light labeling was seen around the border between the ventromedial and the ventrolateral nuclei. At more caudal levels, labeling in the RT was sparse although the dorsal part of the ventromedial nucleus contained a very dense plexus of labeled axons ŽFig. 1C.. Moderate to dense labeling was found in the lateralmost part of the central
Fig. 2. Line drawings showing the site of BDA injection into the ventral part of the RT Žshaded area indicated by a curbed arrow in A. and resulting anterograde labeling in the PF ŽB–D, rostral to caudal.. BDA-labeled fibers and terminals were represented by fine lines and small dots, respectively. APTD, dorsal part of the anterior pretectal nucleus; CPu, caudate putamen; f, fornix; GP, globus pallidus; opt, optic tract. Other abbreviations are as in Fig. 1.
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medial nucleus and in the medialmost parts of the centrolateral and ventrolateral nuclei, whereas labeling was sparse in the paralaminar portion of the mediodorsal nucleus. In the caudal thalamic region, many labeled fibers and terminals were distributed in the ventrolateral part of the PF with a few labeled axons in its ventromedial part ŽFig. 1D, E.. In the second set of experiments, BDA was injected into the ventral part of the RT ŽFig. 2A., where labeled axon terminals had been found after BDA injection into the dorsolateral part of the SNr. Reticular projections were predominantly uncrossed. Dense labeling was found in the ventromedial part of the ventrolateral nucleus, the dorsal part of the ventromedial nucleus and the adjacent parts of the intralaminar nuclei. Light labeling was seen in the ventralmost part of the mediodorsal nucleus. Labeled fibers
with bouton-like varicosities were also distributed in the PF: a moderate to dense plexus of labeled axons was observed not only in the rostralmost part of the PF ŽFig. 2B. but also more caudally in the ventrolateral part of the PF ŽFig. 2C, D.. The plexus extended into the ventromedial part of the posterior thalamic nuclear group, giving rise to light labeling. In the third set of experiments, CTb injection into the ventrolateral part of the PF ŽFigs. 3A and 4A. and BDA injection into the dorsolateral part of the SNr ŽFigs. 3B and 4B. were made on the same side. The distribution pattern of BDA-labeled fibers and terminals in the RT was similar to that in the rats of the first set of experiments. CTb-labeled cell bodies were distributed predominantly in the ventral part of the RT, bilaterally with an ipsilateral dominance. Labeled cell bodies were also found in an area just ventro-
Fig. 3. Line drawings illustrating the sites of CTb injection into the ventrolateral part of the PF Žshaded area in A. and BDA injection into the dorsolateral part of the SNr Žshaded area in B., and resulting distributions of BDA-labeled fibers Žfine lines. and terminals Žsmall dots., and CTb-labeled neurons Žlarge dots. in the RT ŽC–F, rostral to caudal.. Each area enclosed by the small rectangle in the thalamic planes Žc–f. is enlarged in C–F, respectively. MG, medial geniculate nucleus; STh, subthalamic nucleus. Other abbreviations are as in Figs. 1 and 2.
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Fig. 4. Photomicrographs representing the sites of CTb injection into the ventrolateral part of the PF ŽA. and BDA injection into the dorsolateral part of the SNr ŽB., and resulting overlapping distribution of CTb-labeled neurons and BDA-labeled fibers in the ventral part of the RT ŽC,D.. Note bouton-like varicosities labeled with BDA Žarrow heads. tightly surrounding the somata ŽC. and distal dendrites ŽD. of CTb-labeled neurons. Abbreviations are as in Figs. 1 and 3. Bar s1 mm in A,B; 20 mm in C,D.
medial to the rostral portion of the RT, in the lateralmost part of the zona incerta, and within the internal capsule. In the rostral portion of the RT, some labeled cell bodies were seen mainly along the lateral margin ŽFig. 3C.. At the level of the entopedunclular nucleus, the ventral part of the RT contained many labeled cell bodies, most of which were located in the medialmost part or along the lateral margin ŽFig. 3D–F.. More caudally, labeled cells in the RT gradually disappeared. Most of these CTb-labeled neurons were embedded in a plexus of BDA-labeled axon terminals within the ventral part of the RT, where bouton-
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like varicosities labeled with BDA were observed in apposition with cell bodies or proximal dendrites ŽFig. 4C. as well as with distal dendrites ŽFig. 4D. of CTb-labeled neurons. At the electron microscopic level, CTb-labeled neurons were identified by the presence of the electrondense DAB reaction products, which were dispersed not only throughout somatic and large dendritic profiles but also within small dendritic profiles. On the other hand, BDA-labeled fibers and terminal boutons were packed with dense grains of various sizes filling up the cytoplasm except mitochondria. As far as identified, BDA-labeled terminals made symmetrical synaptic contacts with dendrites ŽFig. 5A, B. as well as with soma ŽFig. 5C. of the RT neurons, most of which were labeled with CTb. Out of 48 BDA-labeled terminals forming synaptic contacts with CTb-labeled neurons, which were identified on the photomicrographs, 19 and 29 terminals were in contact with soma and dendrites, respectively. The minor diameter of CTb-labeled dendrites postsynaptic to BDA-labeled terminal boutons ranged from 0.5 to 2.2 mm with a mean of 1.22 mm " 0.37 S.D. The present study indicates that the dorsolateral part of the SNr projects not only monosynaptically but also disynaptically via the ventral part of the RT to the ventrolateral part of the PF. As mentioned in the introduction, the dorsolateral part of the SNr has been considered to contribute to the control mechanism of orofacial movements. Therefore, the nigrothalamic pathways observed here may also be implicated in this mechanism. The nigrothalamic projections from the SNr to the PF have been revealed by using tracer methods w1,4,14x. The present results further showed that the dorsolateral part of the SNr projected to the ventrolateral part of the PF. According to our preliminary study w15x, this part of the PF sends projection fibers to the ventrolateral part of the striatum as well as to the orofacial motor cortex. In addition, the ventrolateral part of the striatum has been known to receive projection fibers from the orofacial motor cortex w23x and to send projection fibers to the dorsolateral part of the SNr w17x. Altogether, these data suggest that within the orofacial motor circuits, the ventrolateral part of the PF is a key station of the thalamic flow to the cerebral cortex and striatum and is under the influence of output neurons of the basal ganglia. An anterograde WGA-HRP study w6x indicated that the SNr sent projection fibers to the ventral part of the rostral two-thirds of the RT. Recently, Kolmac and Mitrofanis w9x reported that neurons in this RT area were retrogradely labeled with biotinylated dextran injected into the PF as well as into other intralaminar nuclei. In the present study, we confirmed these observations by showing the overlapping distribution of the nigral fibers originating from the dorsolateral part of the SNr and the RT neurons projecting to the ventrolateral part of the PF in the ventral part of the RT, and further demonstrated that synaptic connections were made between these nigral fibers and RT neurons.
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Fig. 5. Electron micrographs showing the synaptic connections between the CTb-labeled neurons and the BDA-labeled axon terminals in the ventral part of the RT after combined injections of CTb into the PF and BDA into the SNr. ŽA. Two BDA-labeled terminals Žt1, t2. forming symmetrical synaptic contacts with a dendritic profile Žd1. of the CTb-labeled neuron. A non-labeled dendritic profile Žd2. is also seen. ŽB. Another example of a symmetrical synaptic contact between a BDA-labeled terminal Žt. and a dendritic profile Žd. of the CTb-labeled neuron. ŽC. A BDA-labeled terminal Žt. making a symmetrical synaptic contact with a somatic profile Žs. of the CTb-labeled neuron. Arrowheads indicate symmetrical synaptic specialization. The DAB reaction products are presented by arrows. Bar s 0.5 mm.
The g-aminobutyric acid ŽGABA. neurons in the SNr are well known to send their axons to the thalamus Žsee Ref. w7x for review., and therefore the direct SNr-PF pathway may provide an inhibitory input to PF neurons. Since neurons in the RT are also GABAergic w8,12,13x, the indirect SNr-PF pathway relayed by the RT may exert disinhibitory influence on PF neurons through the inhibitory influence of the SNr on inhibitory RT neurons projecting to the PF. If the SNr exerts its influence simultaneously on PF neurons and PF-projecting RT neurons, PF neurons receiving SNr fibers might be different from those receiving axons of SNr-recipient RT neurons because it seems unlikely that single PF neurons are under the inhibitory and disinhibitory influences of the SNr at the same time. Thus, the direct SNr-PF and indirect SNr-RT-PF pathways, revealed by the present study, may regulate the
thalamic output stream to the cerebral cortex andror striatum by inhibition and disinhibition of PF neurons, respectively, in the control of orofacial movements.
Acknowledgements The authors are grateful for the support of Drs. Ryuzo Fujimoto, Kazuya Fujita, Yasuichi Munenaga, Takashi Sakurai, Yayoi Sakurai, Hiromi Taguchi and Katsumi Tanaka. This study was supported in part by Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan ŽNos. 09832009, 11680740., and by a grant from the Shimane Medical University Education and Research Foundation.
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