Axonal ramification of neurons in the nucleus reticularis tegmenti pontis projecting to the paramedian lobule in the rabbit cerebellum

Neuroscience Research 51 (2005) 15–24 www.elsevier.com/locate/neures

Axonal ramification of neurons in the nucleus reticularis tegmenti pontis projecting to the paramedian lobule in the rabbit cerebellum Dorota Bukowskaa,*, Barbara Mierzejewska-Krzyz˙owskab, Leszek Zguczyn´skib a

Department of Neurobiology, University School of Physical Education, 55 Grunwaldzka St., 60-352 Poznan´, Poland b Department of Anatomy, University School of Physical Education, 13 Estkowskiego St., 66-400 Gorzo´w, Poland Received 13 April 2004; accepted 8 September 2004

Abstract Projections of the nucleus reticularis tegmenti pontis (NRTP) to the cerebellar paramedian lobule were examined in the rabbit by means of the double fluorescent retrograde tract-tracing method. The rabbit NRTP is composed of a medial, large part comprising zones A (dorsomedial), B (central) and C (lateral), and of a lateral, small part (the processus tegmentosus lateralis; PTL). Following unilateral injections of Fast Blue (FB) into the rostral part of the paramedian lobule (rPML) and of Diamidino Yellow (DY) into the caudal part (cPML), known to receive spinal inputs from forelimb and hindlimb, respectively, substantial numbers of single labeled neurons were found in all bilateral NRTP divisions, apart from the zone C. Most projection neurons to the PML were located in the medial and medioventral regions of the zone B. Smaller numbers of projection neurons were located in the PTL, zone A and outside the zone B among fibers of the medial lemniscus. The pattern of FB and DY labeling suggested that neurons projecting to the rPML and cPML originated in common rather than separate regions within the NRTP. In addition, a small percentage (mean 1.3%) of double FB + DY labeled neurons were detected with a clear contralateral preponderance, among single labeled FB or DY cells. In spite of the rarity, all the NRTP neurons giving rise to intralobular collateral projections can be regarded as potential sources of simultaneous modulating influences upon two functional different forelimb (rPML) and hindlimb (cPML) regions. The findings have been discussed in relation to earlier studies in other species and commented on with respect to the possible functional meaning of these projections. # 2004 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved. Keywords: Nucleus reticularis tegmenti pontis; Paramedian lobule; Double labeling; Rabbit

1. Introduction The nucleus reticularis tegmenti pontis (NRTP), considered mainly as the intercalated nucleus in the cerebrocerebellar pathways, receive input from a variety of cortical (Brodal and Brodal, 1971; Brodal, 1980a; Stanton et al., 1988; Shook et al., 1990; Lui and Aldon, 1997; Giolli et al., 2001) and subcortical (Gerrits et al., 1985; Hayakawa and Zyo, 1986; Korp et al., 1989; Noda et al., 1990; Matsuzaki and Kyuhou, 1997; Giolli et al., 2001) areas, and the cerebellar deep nuclei (Schwarz and Schmitz, 1997; Stanton, 2001). * Corresponding author. Tel.: +48 61 835 54 35; fax: +48 61 835 54 44. E-mail address: [email protected] (D. Bukowska).

The NRTP projections are directed exclusively to the cerebellum, both the deep nuclei (Gerrits and Voogd, 1987; Noda et al., 1990; Parenti et al., 2002) and cortex, although projection to the vestibular nuclei is also indicated in the rabbit (Balaban, 1983), but questioned in the cat (Gerrits and Voogd, 1986). The NRTP projections onto the cerebellar cortex including the paramedian lobule (PML) have been demonstrated in the cat (Hoddevik, 1978; Gould, 1980; Kawamura and Hashikawa, 1981; Gerrits and Voogd, 1986), rat (Mihailoff et al., 1981; Herrero et al., 2002; Serapide et al., 2002a), rabbit (Grottel et al., 1988, 1989) and monkey (Brodal, 1980b, 1982). It is generally accepted that the NRTP projections supply almost all parts of the cerebellar cortex (with the exception of vermal lobule X) and show bilateral, but mainly contralateral preponderance.

0168-0102/$ – see front matter # 2004 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved. doi:10.1016/j.neures.2004.09.011

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The pontocerebellar mossy fibers, both originating from the basilar pontine nuclei (Mihailoff, 1983; Rosina and Provini, 1984; Bukowska et al., 2003) and the NRTP, are collateralized. There are only few reports describing collateral projections from the NRTP. In the rabbit, branchings of NRTP axons to the PML originate bilaterally from defined regions of the NRTP (Mierzejewska-Krzyz˙ owska, 1999). In the rat, interhemispheric branches of NRTP axons distribute to pairs of homotopic (corresponding) and heterotopic (non-corresponding) lobules (Mihailoff, 1983; Payne, 1983) and to heterotopic lobules within the same hemisphere (Mihailoff, 1983). Also, scarce intralobular branching projections to crus II-anterior and crus II-posterior of the ansiform lobule were reported in the rat (Mihailoff, 1983). In this context, it would be interesting to study whether the NRTP neurons may participate in the intralobular branchings to supply other cerebellar regions. The functional organization of most of the cerebellar cortical areas that receive branching projections from the NRTP is poorly understood. The PML, a major part of the intermediate zone of the cerebellar posterior lobe involved in the control of ipsilateral limb movements (Ito, 1984; Kandel et al., 2000) exhibits a clearly defined somatotopical organization. It has been proved, mainly with electrophysiological methods, that the rostral part of PML (rPML) is supplied by afferent fibers from the forelimb and face regions, whereas the more caudal PML (cPML) is influenced by fibers from the hindlimb (Cooke et al., 1972; Inui, 1989; Atkins and Apps, 1997). With regard to the intrinsic somatotopy of the PML, the present study was undertaken to investigate whether the NRTP neurons project to the rPML (forelimb) and cPML (hindlimb) independently or whether their axons may bifurcate to supply simultaneously these two non-homologous PML regions. To answer this question, the double fluorescent retrograde technique was employed in the rabbit.

2. Material and methods The research reported herein was performed according to the Polish Law on Animal Protection and under guidelines established by the Declaration of Helsinki concerning the appropriate Care and Use of Animals in Research. The experiments were carried out on nine adult New Zealand white rabbits (five male and four female) weighing between 2.0 and 3.5 kg. All surgical procedures were performed aseptically under deep anesthesia by using a mixture of ketamine hydrochloride (Calypsol, 50 mg/kg BW) and promazine (19 mg/kg BW) at 1:0.38 ratio injected intramuscularly. Supplemental doses were given as necessary to maintain anesthesia. After the head of animal was fixed in a stereotaxic holder (Narishige), two small craniotomies were made to expose the dorsal surface of

the rPML and cPML sublobules on the right side. Then, under control of an operating microscope they were pressure-injected in several (three to five) points (total 1.8–2.6 ml) with 4–5% Fast Blue (FB) and 2% Diamidino Yellow (DY), respectively, using a glass micropipette (inner tip diameter of 30–60 mm) secured on a 5 ml Hamilton microsyringe. Both tracers were sonicated (FB for 5 min and DY for 15 min) shortly before use to improve uptake and retrograde transport by axons to parent perikarya. In order to avoid undesired leaking of the tracer outside the injection site, the micropipette was left in place for 5–10 min before and after injection. After injections were completed, the craniotomies were packed with gel foam and the muscle and skin were closed with sutures. The animals were allowed to survive for 9–15 days. Then, they were deeply re-anesthetized and sacrificed by transcardiac perfusion with about 1.5–2.0 l of 0.9% saline rinsing solution in phosphate buffer (PB; pH 7.4) mixed with heparin at body temperature, followed immediately by about 1.5 l of fixative at room temperature containing 20% formalin solution in 0.4 M PB (pH 7.4) and then by 1.0 l of cold (4 8C) 10% sucrose–PB solution. After the perfusion, the skull was opened and the brain removed, and the approximate location of the injection sites in the superficial region of the PML sublobules was determined by macroscopic inspection. Then, the brain was cut into two blocks (brainstem and cerebellum) and stored overnight in 20% sucrose–PB solution (4 8C) prior to sectioning for cryoprotection. The pons was cut transversely and the cerebellum sagittally with the use of a freezing microtome (Reichert, Austria) at 40-mm thick serial sections that were consecutively collected in dishes containing 0.1 M PB. Two out of three sections were mounted on chrom-alumgelatinized glass slides, air-dried and then dehydrated, cleared in xylene and coverslipped with Fluoromont (Serva). All mounted sections were examined using the Optiphot2 (Nikon, Japan) and Jenalumar (Carl Zeiss Jena, Germany) microscopes equipped with epi-fluorescence illumination (ultraviolet filter, 380 and 410 nm light excitation wavelength, respectively) (Keizer et al., 1983). The extent of the injection site was determined under a 10 times magnifying objective and superimposed onto a diagram of reconstruction of the unfolded cerebellar cortex of the rabbit adopted from Brodal (1940) and onto diagrams of four sagittal sections of the right PML drawn from Nissl stained sections of a normal rabbit. According to Kuypers et al. (1980) and Keizer et al. (1983), labeled FB or DY neurons exhibited blue fluorescence in the neuroplasm or yellow fluorescence in the nucleus, respectively. Double FB + DY labeled neurons were recognized by simultaneous fluorescence of both tracers. In each case, both single and double labeled neurons were counted in two out of three mounted serial sections throughout the entire rostrocaudal extent of the NRTP (average 3040 mm). Thus, in each individual experiment with 40-mm thick sections, labeled neurons

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could be counted in approximately 51 out of 76 sections. To facilitate the comparison of findings between different experiments, the distribution of labeled neurons was demonstrated on diagrams of eight equally spaced (390mm intervals) transverse sections through the rostrocaudal extent of the NRTP.

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3. Results 3.1. Anatomy of the PML The rabbit PML is composed of six sublobules, from caudal ‘‘a’’ to rostral ‘‘f’’ following the nomenclature of

Fig. 1. Diagrams illustrating the pairs of FB (vertical hatching) and DY (horizontal hatching) injection sites, including diffusion zone, in the rPML and cPML sublobules. Upper panel shows the cerebellar dorsal surface of the right side represented as unfolded in one plane and the bottom panel shows four sagittal sections through the PML, in representative cases Ra1, Ra3, Ra5, Ra6 and Ra7. Vertical lines numbered 1–4 (upper) indicate the planes which correspond to the planes of sagittal sections (bottom). c, l, m, r, caudal, lateral, medial, rostral directions. a–f: PML sublobules from caudal to rostral. An: ansiform lobule. Cp: copula pyramidis. fp: primary fissure. HV: fifth lobule of hemisphere. Pf: paraflocculus. Ve: vermis. Scale bar for bottom diagrams is 5 mm.

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Fig. 2. Photomicrographs of sagittal sections through injection sites in the PML sublobules and resulting retrograde labeling of neurons in the NRTP showed on transverse sections. (A) FB deposit in sublobule f of the rPML in case Ra4. (B) DY deposit in sublobule b of the cPML in case Ra5. This section corresponds to that between 2 and 3 in Fig. 1. Superficial tissue damage because of multiple penetrations of micropipette during injection is seen. (C–G) Single and double labeled neurons in: (C) ipsilateral PTL between levels Vand VI in case Ra5; (D) contralateral zone B at level VII in case Ra3; (E) contralateral zone B at level VI in case Ra3; (F) ipsilateral zone A at level IV in case Ra9; and (G) ipsilateral Cb at level V in case Ra9. Note blue fluorescence in the neuroplasm and dendrites in FB single labeled neurons (large asterisk) and yellow (brilliant) fluorescence in the nucleus in DY single labeled neurons (small asterisk), and FB + DY double labeled neurons (arrow) displaying both nuclear (DY) and neuroplasmic (FB) fluorescence. Scale bar = 600 mm for (A), 450 mm for (B), 100 mm for (D), 60 mm for (E), 30 mm for (G), 20 mm for (C) and (F).

348 Total after all injections Mean

In each rabbit, the number of labeled neurons was counted in two out of three serial sections of NRTP; percentage (%) of the total number of double labeled neurons was counted in relation to the total number of single and double labeled neurons.

0.5 0.4 7 1 2 5 1335 148 820 515 2.5 2.6 17 2 10 7 655 73 501 154 1.1 1.2 146 16 94 52 13718 1524 7860 5858 2.1 0.9 16 2 10 6 745 83

i %

0.9 – – – – – 2.2 1.8 3.4 1 – – – – – 2 4 9

Total

1 – – – – – 1 3 5 – – – – – – 1 1 4

c Total

115 8 – 13 14 36 89 214 256 39 6 – 13 11 19 41 132 136

c

397

– 0.9 – – 0.9 – – 0.9 0.5 – 1 – – 3 – – 2 1

Total c

– – – – 1 – – 1 – – 1 – – 2 – – 1 1

i Total

227 108 – 29 340 182 29 216 204 121 67 – 29 189 89 – 193 132

c i

106 41 – – 151 93 29 23 72 – 1.0 – 2.3 10 – 3.9 3.2 3.4

% Total

– – – – 2 – 2 2 4 c – 1 – 1 – – 1 – 4

i Total

55 98 42 42 18 36 74 61 229 32 87 25 30 14 23 56 44 190

c

23 11 17 12 4 13 18 17 39 i %

1.2 1.0 2.5 1.3 0.9 0.2 1.6 0.5 1.4 22 12 17 14 19 3 19 11 29

Total c

17 12 11 7 10 – 14 6 17 5 – 6 7 9 3 5 5 12

i Total

1870 1237 667 1047 2032 1563 1193 2091 2018 929 659 426 609 1138 997 715 1144 1243

c i i

941 578 241 438 894 566 478 947 775

Single

76 2 – – 3 17 48 82 120

Double PTL

Single Double

Cb

Single Double

B

Single labeled neurons were found bilaterally in all NRTP divisions through its rostrocaudal extent (numerously in the central part), apart from the zone C where no labeling could be observed (Table 1, Figs. 3 and 4). The ratio of contra/ ipsilateral cells in all rabbits was 58% contra- and 42% ipsilateral. Labeled neurons were primarily found in the medial and medioventral regions of zone B, but also ventrally. In the PTL, they were present through entire mediolateral extent with great density in the midlateral regions. The smallest number of labeled neurons was found in the medial and medioventral regions of zone A. In addition, labeled

Double

3.4. Distribution of single labeled neurons

Single

All injections were made just below the dorsal surface of the PML sublobules encroaching all three layers of cortex. In most cases, diffusion of tracers was also visible in the underlying white matter, but only in the apical part of the sublobule stem. Several cases where the tracer accidentally spread deeper into white matter were excluded from analysis in the present study. The rostrocaudal and mediolateral extents of injection sites in the rPML and cPML differed more or less in individual cases as it was shown in Figs. 1 and 2A, B. In no experiment was there any overlap between the two injection sites, including the diffusion zone, and no contamination with injected tracer was observed in the adjoining ansiform lobule or vermis.

A

3.3. Injection sites in the PML

Case number

The rabbit NRTP is situated ventrally to the central superior nucleus, ventromedially to the nucleus reticularis pontis oralis and caudalis, and dorsally to the pontine gray proper (basilar pontine nuclei) separated from the latter by the medial lemniscus. In comparison to the reticular formation located dorsally, the NRTP neurons are densely packed and aggregated in clusters. The NRTP consists of two main parts, a medial, larger part which corresponds to the nucleus papilliformis described by Meessen and Olszewski (1949) and a smaller, lateral extension termed the processus tegmentosus lateralis (PTL) by Brodal and Brodal (1971). Within the medial part, three zones can be discerned on the basis of clustering of cells segregated by white matter and differences in size of soma: zone A in the dorsomedial region (levels III–VII), zone B in the main, central region (levels I– VIII) and zone C in the smaller, lateral region of this nucleus (levels VI–VII) (Grottel et al., 1988). In addition, neurons outside the medioventral border of zone B form the cell bridges (Cb) extending within fiber bundles of the medial lemniscus toward the basilar pontine nuclei.

Table 1 Number of single and double labeled neurons in the ipsilateral (i) and contralateral (c) NRTP divisions as a result of FB and DY injections into the rostral and caudal PML sublobules

3.2. Nomenclature and subdivisions of the NRTP

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Ra1 Ra2 Ra3 Ra4 Ra5 Ra6 Ra7 Ra8 Ra9

Brodal (1940). We refer to sublobules a, b and c as the caudal part of PML, and to sublobules d, e, and f as the rostral part of PML.

– 1 – 1 2 – 3 2 8

%

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neurons appeared outside the medioventral border of zone B, in the Cb, invading fibers of the medial lemniscus. The NRTP labeled neurons had soma diameters ranging from 15 to 35 mm and shapes from oval to triangular or polygonal. Whereas oval-shaped somata predominated in all NRTP divisions, their size was apparently the largest in the zone A, and medium in the zone B. Neurons in the PTL exhibited the smallest size, however some fairly large perikarya could be also seen scattered in this division (Fig. 2C–E). The NRTP neurons labeled with FB or DY were not composed of segregated populations, but were intermingled in common areas. Thus, no topographical relationships were observed between injection sites and the location of labeled neurons.

3.5. Distribution of double labeled neurons There were fewer double labeled neurons in the NRTP (n = 186), but their distribution was the same as the neurons labeled with either FB or DY. Such neurons were found in the zones B (n = 146) and A (n = 16) clustering in the medial and medioventral regions (Fig. 2D–F), but were absent in the ventral region of zone B. They appeared also in the lateral region of the PTL (n = 7) and in the Cb (n = 17) (Fig. 2C and G). Double labeled neurons in the NRTP were distributed bilaterally, with a greater number on the contralateral side (62% contra- versus 38% ipsilaterally). These neurons reflected sizes and shapes of those single labeled with FB as it has been described above in the particular NRTP divisions.

Fig. 3. Diagram of transverse sections through the ventral regions of the pons showing distribution of FB (white triangles) and DY (white squares) single labeled, and of FB + DY (black dots) double labeled neurons in the NRTP divisions resulted from injection of tracers into the rPML and cPML, in case Ra5. Each white triangle and square denote approximately 20 single labeled neurons, apart from the zone A, where one triangle denotes about 5 labeled neurons. Black dots indicate exact number of double labeled neurons (n = 24). Sections are spaced with equal (about 390 mm) intervals. ML: medial lemniscus. PN: pontine nuclei. Py: pyramis. TB: nucleus of trapezoid body. Scale bar = 900 mm.

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Fig. 4. Summarizing diagram showing the areas occupied by single FB (vertical hatching) and DY (horizontal hatching) labeled neurons on transverse sections through the NRTP divisions, i.e., neurons projecting to the rPML and cPML sublobules, respectively. These areas are demarcated on the basis of individual distribution of FB and DY single labeled neurons in all nine rabbits. The common areas for the neurons are cross-hatched. All these areas reflect the largest extents of labeled neurons, but not their density. Sections are spaced with equal (about 390 mm) intervals. Scale bar = 900 mm.

Fig. 5. Summarizing diagram showing distribution of FB + DY double labeled neurons (n = 186) on transverse sections through the NRTP divisions, recognized in the present material from all nine rabbits. These neurons represent the cells of origin for collateral projection to the rPML (forelimb-face) and cPML (hindlimb). One dot represents one double labeled cell body. Sections are spaced with equal (about 390 mm) intervals. Scale bar = 900 mm.

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Thus, there appeared to be no obvious relationship between the size and shape of perikaryon and whether it was single or double labeled. Considering the number of both single and double labeled neurons, those with FB + DY tracers constituted on an average 0.9% in zone A (0–9 neurons per rabbit), 1.2% in zone B (3–29 neurons per rabbit), 2.6% in the Cb (0–8 neurons per rabbit) and 0.4% in the PTL (0–3 neurons per rabbit). The distribution of double labeled neurons together with single labeled neurons is shown in Fig. 3 (case Ra5) and as separate plot of all cases in Fig. 5.

4. Discussion 4.1. Origin and organization of the NRTP-PML projections The present study on rabbits demonstrates that the PML receives afferents from a substantial number of neurons located primarily in the medial and medioventral regions of the main NRTP part, the Cb, and from the entire PTL. The data indicate that the projection is bilateral, with a small contralateral predominance (58% crossed versus 42% unilateral fibers). The same finding concerning laterality was obtained in the rat (Serapide et al., 2002b), but differed in the monkey where almost all NRTP-PML fibers were crossed (Brodal, 1980b, 1982). The results presented herein are in general in agreement with those of previous anatomical studies in the rabbit with the use of horseradish peroxidase (HRP) as a retrograde marker (Grottel et al., 1988, 1989). Some minor differences in projections from the zone C and the PTL may have been due to differences in type of tracers used in the two studies. The present findings are, in broad terms, conformable to those obtained in the cat (Hoddevik, 1978; Gould, 1980) and rat (Mihailoff et al., 1981) where the NRTP-PML projections originate from the medial part of the NRTP. This NRTP region in the cat and rat seems to be homologous with the medial region of zone B in the present material. Our results also agree with those from an anterograde tracing study in the cat where injections of 3H amino acids in the medial regions of NRTP resulted in dense labeling of axon terminals in the PML (Kawamura and Hashikawa, 1981; Gerrits and Voogd, 1986) but injections in the dorsomedial parts of NRTP resulted in labeling of only a few axon terminals there (Kawamura and Hashikawa, 1981). Moreover, projection from the NRTP to the PML has been demonstrated by the use of biotinylated dextran amine injected into discrete regions of the NRTP as terminating in two sagittal zones in the rat (Serapide et al., 2002a). The present material is not a suitable source to comment on the issue of zonal organization because the injections are too large to reveal details in projection pattern of small clusters of the NRTP neurons.

4.2. Axonal branching of the NRTP-PML projecting neurons Collateral projections of the pontocerebellar neurons have been observed as originating from the basilar pontine nuclei (Mihailoff, 1983; Rosina and Provini, 1984; Bukowska et al., 2003) and the NRTP (Mihailoff, 1983; Payne, 1983; Mierzejewska-Krzyz˙ owska, 1999) in several animal species. Interhemispheric divergence of axons from the NRTP has been shown to terminate in the pairs of homotopic (lobulus ansiformis, lobulus simplex and the PML) and heterotopic (the PML and crus II, and lobulus simplex and crus II) lobules in the rat (Mihailoff, 1983; Payne, 1983) and in the PML of both sides in the rabbit (Mierzejewska-Krzyz˙ owska, 1999). Moreover, intrahemispheric collateral projections to the PML and crus II (interlobular), and to the crus II-anterior and crus IIposterior of the ansiform lobule (intralobular) were found in the rat ((Mihailoff, 1983). A new observation in this study is the presence of a small but consistent population of double labeled neurons in the NRTP which represents the parent neurons for projection of divergent axons to the rostral and caudal parts of the PML. These neurons do not constitute separate groups but are present only in the regions of the overlap of single labeled cells. About 1.3% of the total population of labeled neurons in the NRTP that project to the PML contribute to collateral projections. Such neurons can be regarded as potential sources of simultaneous modulating influences upon neurons of two functional different forelimb (rPML) and hindlimb (cPML) regions. 4.3. Functional implications A role of the medial NRTP is the mediation and processing of optokinetic signals which are directed to the posterior vermal lobules VI and VII and flocculus, regarded as visual-related and eye movement-related regions (Miles and Fuller, 1975; Lisberger and Fuchs, 1978; Suzuki and Keller, 1988). In the present study, at least some of the neurons projecting to the PML seem to distribute within the NRTP region that contains neurons that project bilaterally to the flocculus in the rabbit (Maekawa et al., 1981), cat (Gerrits and Voogd, 1986), rat (Osanai et al., 1999) and monkey (Nagao et al., 1997) and to the vermal visual area in rabbit (Alley et al., 1975), cat (Matsuzaki and Kyuhou, 1997), rat (Pa¨ a¨ llysaho et al., 1991) and monkey (Yamada and Noda, 1987). It appears from the above reports that general principles of the anatomical organization of the NRTP projections onto the cerebellar cortex are very similar among species, although there are differences in the visual organization of the nucleus between species with frontally and laterally placed eyes. In the monkey (foveate animal), for example, the medial region of the NRTP receives its major cortical input from the frontal eye field (Stanton et al., 1988) and

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supplementary eye field (Shook et al., 1990) shown to be involved in smooth-pursuit and saccadic eye movements (Yamada et al., 1996; Suzuki et al., 2003) or in vergence and accommodative eye movements (Gamlin and Clarke, 1995). Studies on the rabbit (afoveate animal) showed that the NRTP is influenced by the frontal cortex (Abdel-Kader, 1968; degeneration method) and emphasized importance of this nucleus for horizontal optokinetic eye movements (Miyashita and Nagao, 1984; Kano et al., 1991). Apart from cortical inputs, the NRTP is the recipient of afferents from the nucleus of the optic tract (rabbit, Maekawa and Kimura, 1981; rat, Korp et al., 1989), superior colliculus (monkey, Scudder et al., 1996; cat, Matsuzaki and Kyuhou, 1997), vestibular nuclei (rabbit, Balaban, 1983; cat, Gerrits et al., 1985) and cerebellar deep nuclei (rat, Schwarz and Schmitz, 1997; monkey, Stanton, 2001). In the rabbit, subcortical projections to the medial region of NRTP were reported to originate from diencephalic limbic and other areas such as mamillary and habenular nuclei, nucleus of the Forel field, pretectum, zona incerta, superior colliculus, vestibular and prepositus hypoglossi nuclei (Hayakawa and Zyo, 1986). It seems likely that the projections from the medial NRTP to the rPML and cPML, described in the present study might relay signals from above mentioned centers to participate in the coordination of the forelimb– hindlimb muscles with simultaneous adjustment of eye and head positions. Electrophysiological studies using micromapping techniques revealed that peripheral projections to the cerebellar cortex exhibit patchy-mosaic features. The fractured somatotopic maps of the PML have been described in the cat, rat, opossum and galago (Shambes et al., 1978; Robertson, 1984; Welker, 1987; Welker et al., 1988). They have similar cortical pattern, but differ in the proportional representation and location of various body surfaces that may reflect morphological and behavioral differences between these species. It can be assumed that the multiple patches and general arrangement of the body surface representation are also similar in the rabbit. However, it is difficult to say which patches are selected by the NRTP neurons showed herein as projecting to the rPML or cPML, or to both rPML and cPML. It is not unlikely also that NRTP afferents to the PML select cortical patches related to proximal limb muscle groups regardless of the forelimb or hindlimb, because the PML is a component of the cerebellar intermediate zone that control affects primarily the proximal parts of limb, rather than the wrist and digits (Kandel et al., 2000). The present findings provide anatomical evidence of axonal collateral projections from the NRTP neurons to the rPML and cPML, and in order to understand functional meaning, they should be extended with the use of electrophysiological and immunocytochemical procedures to identify the types of inputs on these neurons and neurotransmitter used in this pathway.

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