Spinal projections from the presumptive midbrain ...

Brain Struct Funct (2012) 217:211–219 DOI 10.1007/s00429-011-0337-6

ORIGINAL ARTICLE

Spinal projections from the presumptive midbrain locomotor region in the mouse Huazheng Liang • George Paxinos Charles Watson

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Received: 7 March 2011 / Accepted: 23 June 2011 / Published online: 7 July 2011 Ó Springer-Verlag 2011

Abstract The mesencephalic locomotor region (MLR) plays an important role in the control of locomotion, but there is ongoing debate about the anatomy of its connections with the spinal cord. In this study, we have examined the spinal projections of the mouse precuneiform nucleus (PrCnF), which lies within the boundaries of the presumptive MLR. We used both retrograde and anterograde labeling techniques. Small clusters of labeled neurons were seen in the medial portion of the PrCnF following fluorogold injections in the upper cervical spinal cord. Fewer labeled neurons were seen in the PrCnF after upper thoracic injections. Following the injection of anterograde tracer (biotinylated dextran amine) into the PrCnF, labeled fibers were clearly observed in the spinal cord. These fibers traveled in the ventral and lateral funiculi, and terminated mainly in the medial portions of laminae 7, 8, and 9, as well as area 10, with an ipsilateral predominance. Our observations indicate that projections from the PrCnF to the spinal cord may provide an anatomical substrate for the role of the MLR in locomotion. Keywords Fluoro-gold Spinal cord Biotinylated dextran amine Precuneiform nucleus H. Liang G. Paxinos C. Watson Neuroscience Research Australia, Randwick, NSW 2031, Australia H. Liang G. Paxinos School of Medical Science, The University of New South Wales, Sydney, NSW 2052, Australia C. Watson (&) Faculty of Health Sciences, Curtin University, GPO Box U1987, Shenton Park Health Research Campus, Perth, WA 6845, Australia e-mail: [email protected]

Mesencephalic locomotor region Retrograde labeling Anterograde labeling Abbreviations 3Sp Lamina 3 of the spinal gray 4Sp Lamina 4 of the spinal gray 5SpL Lamina 5 of the spinal gray, lateral part 5SpM Lamina 5 of the spinal gray, medial part 6SpL Lamina 6 of the spinal gray, lateral part 6SpM Lamina 6 of the spinal gray, medial part 7Sp Lamina 7 of the spinal gray 8Sp Lamina 8 of the spinal gray 10Sp Area 10 of the spinal gray Aq Aqueduct Ax9 Axial muscle motoneurons of lamina 9 BDA Biotinylated dextran amine Bi9 Biceps motoneurons of lamina 9 CC Central canal CeCv Central cervical nucleus CEx9 Crural extensor motoneurons of lamina 9 CFl9 Crural flexor motoneurons of lamina 9 CnF Cuneiform nucleus DAB 3,30 -Diaminobenzidine De9 Deltoid muscle motoneurons of lamina 9 FEx9 Forearm extensor motoneurons of lamina 9 FFl9 Forearm flexor motoneurons of lamina 9 Gl9 Gluteal motoneurons of lamina 9 Hm9 Hamstring motoneurons of lamina 9 IB Internal basilar nucleus ICl Intercalated nucleus ICo9 Intercostals muscle motoneurons of lamina 9 IH9 Infrahyoid muscle motoneurons of lamina 9 IML Intermediolateral column IMM Intermediomedial column KF Kolliker-fuse nucleus

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LatC LD9 LPAG LSp M5 MLR mRt PAG PB Pec9 Ph9 PL Pn PnO PrCnF PTg Rh9 RtTg SC SI9 Sr9 SubCV Tr9 TzSM9 VLPAG WGA-HRP

Brain Struct Funct (2012) 217:211–219

Lateral cervical nucleus Latissimus dorsi motoneurons of lamina 9 Lateral periaqueductal gray Lateral spinal nucleus Motor trigeminal nucleus Mesencephalic locomotor region Mesencephalic reticular formation Paeriaqueductal gray Phosphate buffer Pectoral muscle motoneurons of lamina 9 Phrenic motoneurons of lamina 9 Paralemniscal nucleus Pontine nuclei Oral part of pontine reticular nucleus Precuneiform nucleus Peduncular tegmental nucleus Rhomboid muscle motoneurons of lamina 9 Reticulotegmental nucleus of the pons Superior colliculus Supraspinatus and infraspinatus motoneurons of lamina 9 Serratus anterior motoneurons in lamina 9 Ventral part of the subcoeruleus nucleus Triceps motoneurons of lamina 9 Trap/sternom motoneurons of lamina 9 Ventrolateral periaqueductal gray Wheat germ agglutinin-horseradish peroxidase

Introduction The electrical stimulation of the mesencephalic locomotor region (MLR) evokes rhythmic stepping behaviour in decerebrate animals (Garcia-Rill et al. 1983; Skinner and Garcia-Rill 1984; Atsuta et al. 1990; Bernau et al. 1991), indicating that this region plays a role in the generation of locomotor patterns. The MLR region is generally considered to lie deep to the superior colliculus (SC) and lateral to the periaqueductal gray (PAG) (Garcia-Rill 1983). A number of distinct nuclear groups occupy this region, any or all of which might give rise to spinal connections that could initiate locomotor activity. The candidate nuclei in this area include the cuneiform nucleus (CnF) (Garcia-Rill et al. 1983; Skinner and Garcia-Rill 1984; Coles et al. 1989; Allen et al. 1996), the precuneiform nucleus (PrCnF) (Liang et al. 2011), the mesencephalic reticular formation (mRt) (Garcia-Rill et al. 1985), and the peduncular tegmental nucleus (PTg) (Skinner and Garcia-Rill 1984; Garcia-Rill et al. 1987; Coles et al. 1989). We have assumed that the position of the MLR in the mouse is

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homologous with the region identified in the cat, and have studied the spinal projections that arise from this region of the midbrain. In a previous retrograde tracing study in the mouse, we showed that cells in the PrCnF, the mRt, and the PTg projected to the spinal cord. We did not find labeled cells in the CnF (except for a small number of cells in its most rostral part) (Liang et al. 2011). We consider it unlikely that the PTg is an effector nucleus for the MLR because it is too deep and too caudal to be stimulated in such physiological experiments. It must be noted that the SC and the PAG have also been shown in many species to project to the spinal cord (Altman and Carpenter 1961; Nyberg-Hansen 1964; Martin 1969; Kuypers and Maisky 1975; Graham 1977; Harting 1977; Castiglioni et al. 1978; Basbaum and Fields 1979; Nudo and Masterton 1988; Masson et al. 1991; Cowie and Holstege 1992; Satoda et al. 2002; VanderHorst and Ulfhake 2006; Liang et al. 2011), but neither of these areas have been implicated in the initation of locomotion (Bernau et al. 1991). We conclude that the PrCnF and the mRt are the centers most likely to connect the MLR with the spinal cord. The identity of the PrCnF is largely based on distinguishing it from other better known cell groups in the midbrain. However, it can be differentiated from its neighbours on the basis of SMI32 immunohistochemical preparations in the mouse (Watson and Paxinos 2010, Figs. 158–183). In the mouse, the PrCnF extends from the level of the caudal pole of the red nucleus to the level of the rostral pole of the motor trigeminal nucleus (M5). It is located ventral to the SC, lateral to the PAG, and dorsal to the mRt (Franklin and Paxinos 2008). Lateral to the PrCnF are a series of auditory structures: there are, from rostral to caudal, the external cortex of the inferior colliculus and the lateral lemniscus. The caudal pole of the PrCnF lies dorsal to the CnF, which has distinctive horizontal band of acetylcholine esterase. The medial border of the PrCnF is formed by the distinctive boundary of the PAG. Immunohistochemical study shows that it has scattered parvalbumin, calbindin, and calretinin positive neurons (Paxinos et al. 2009a). In this study, we have examined the spinal projections of the PrCnF with retrograde and anterograde tracing methods. We have shown that cells in the PrCnF send their axons to medial areas of the lamina 7 interneuron pool in cervical and upper thoracic segments, and might therefore be an effector pathway for the MLR. A further issue that must be considered is the definition of nuclei that lie within the presumpitve MLR. There are different opinions concerning the boundaries of the CnF, PrCnF, and the mRt in different species (Panneton and Watson 1991; Swanson 1998; Paxinos and Watson 2007; Franklin and Paxinos 2008; Paxinos et al. 2009b). This may explain why studies in cats and monkeys found plentiful spinal projections from the CnF (Castiglioni et al.


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Forty C57BL/6 mice (10–12 weeks old, with a weight of 25–30 g) were used for this study. The mice were obtained from the Animal Resource Center in Western Australia. The experimental procedures were approved by the Animal Care and Ethics Committee of the University of New South Wales (08/48B).

Science Tools, North Vancouver, BC, Canada). The PrCnF was injected with 10–20 nl of biotinylated dextran amine (BDA) solution (10,000 MW, Invitrogen, Mulgrave, VIC, Australia) (Bregma: -4.16 to -4.72 mm, midline: ?1.0 to ?1.25 mm, surface: -2.175 to -2.5 mm; 6 mice) using the same Hamilton syringe as for retrograde tracer injections. Control animals received the same tracer injections either into the cisterna magna (3 mice) or into the adjacent brain areas surrounding the PrCnF. In these cases, BDA was deliberately injected to the adjacent PAG, SC, and mRt in a total of 11 mice in order to control for spread of tracer from PrCnF injections. In each case, the syringe was left in place for 10 min after the injection. At the end of the procedure, the soft tissue and the skin were sutured, buprenorphine was injected subcutaneously, and topical tetracycline was sprayed over the incision.

Retrograde tracing

Tissue preparation

Twenty mice were used for the retrograde study. They were anaesthetised with an intraperitoneal injection of ketamine (67 mg/kg) and xylazine (10 mg/kg), before they were mounted in a mouse stereotaxic head holder (Kopf Instruments, Tujunga, CA, USA). A 5 ll Hamilton syringe (Hamilton Company, Reno, NV, USA) was mounted on a micromanipulator for spinal cord injection. The mouse adaptor was adjusted for optimal exposure of the vertebrae. Upper cervical, upper thoracic, and upper lumbar spinal cord segments were exposed by laminectomy at C2, T3– T4, and T11–T12, respectively. The dura on the right side was incised with the tip of a 29-gauge needle and the 5 ll Hamilton syringe was driven through this opening. The outer diameter of the Hamilton syringe was 0.711 mm. An injection of 20–40 nl of fluoro-gold (Fluorochrome, Denver, CO, USA) diluted to 5% in distilled water was made through multiple punctures into the right side of the spinal cord. The syringe was left in place for 10 min following the injections. Altogether, 15 mice were injected with fluoro-gold to the upper cervical, upper thoracic, and upper lumbar segments (5 mice in each group). The control group either received normal saline injections into the spinal cord (2 mice) or fluoro-gold injections into the cisterna magna (3 mice). At the end of the procedure, the soft tissue and the skin were sutured and topical tetracycline (Pfizer) was sprayed over the incision. To relieve the postoperative pain, subcutaneous buprenorphine (Tamgesic, Reckitt Benckiser) injections were applied.

After survival times of 96 h (flouro-gold experiments) or 6 weeks (dextran experiments), the mice were anesthetised with a lethal dose of pentobarbitone sodium (0.1 ml, 200 mg/ml) and perfused through the left ventricle with 60 ml of 0.9% normal saline containing heparin (150 IU/ mouse; Sigma, Castle Hill, NSW, Australia). This was followed by 80 ml of 4% paraformaldehyde (Sigma) prepared in 0.1 M phosphate buffer (PB: Na2HPO4, 0.1 M; NaH2PO4, 0.1 M; pH = 7.4) and finally with 80 ml of 10% sucrose solution. The brain and spinal cord were removed and postfixed in 4% paraformaldehyde for two hours at 4°C, followed by cryoprotection in 30% sucrose in 0.1 M PB solution overnight at 4°C. Serial sections of the brain and spinal cord were cut at 40 lm using a Leica CM 1950 cryostat. Sections from the fluoro-gold injected mouse spinal cords were mounted onto gelatinized slides and coverslipped with an anti-fade fluorescent mounting medium (Dako, Campbellfield, VIC, Australia). The tissue sections were examined with a Nikon Eclipse 80i fluorescence microscope.

1978; Satoda et al. 2002), while we found very few in the mouse (Liang et al. 2011). It is probable that the size of the PrCnF in the monkey and cat has not been fully appreciated (Castiglioni et al. 1978; Satoda et al. 2002).

Materials and methods Animals

Anterograde tracing Twenty mice were anaesthetised as described above and mounted in the stereotaxic head holder. After incising the skin, the skull was penetrated with a bone drill (Fine

Immunohistochemistry The brain sections from the fluoro-gold injected mice were washed in 0.1 M PB and treated with 1% H2O2 in 50% ethanol for 30 min at room temperature. The sections were rinsed in 0.1 M PB and then treated with 5% goat serum in 0.1 M PB to block the non-specific sites. The sections were incubated with the primary anti-fluoro-gold antibody (Chemicon, 1:2,000; raised in rabbit) overnight, rinsed in 0.1 M PB (39), then treated with the secondary antibody (biotinylated goat anti-rabbit IgG; Sigma, 1:200) for 2 h at room temperature. The sections were then washed in 0.1 M PB, and transferred to an extravidin peroxidase solution

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(Sigma, 1:1,000) for 2 h. After a rinse in 0.1 M PB, the sections were transferred to the 3, 30 -diaminobenzidine (DAB) reaction complex (Vector lab, Burlingame, CA, USA) until optimal color developed. At the end of the procedure, the sections were rinsed, mounted onto gelatinized slides, dehydrated in gradient ethanol, cleared in xylene, and coverslipped. The sections from the dextran injected mouse brains and spinal cords were treated with 1% H2O2 in 50% ethanol for 30 min at room temperature, followed by 5% goat serum in 0.1 M PB for 30 min. Then the sections were transferred to an extravidin peroxidase solution (Sigma, 1:1,000) for 2 h. The following procedures were the same as for the fluoro-gold immunostain. Data analysis The sections from fluoro-gold injected mouse brains were mapped using the Stereoinvestigator software (MicroBrightfield, Williston, VT, USA). The images were taken by an Optronics camera (Goleta, CA, USA) mounted onto a Nikon Eclipse 80i microscope, which was connected to a Dell Precision T3500 workstation. Reference to adjacent Nissl stained sections assisted in this mapping process. First, the contour was drawn at 29 magnification, and then the position of each labeled neuron was marked at 109 magnification. Cell numbers were automatically calculated by counting the marked cells in every second section through the entire PrCnF. For cell counting, three series of sections (prepared from 3 mice) were employed. Only labeled neurons with a visible nucleus or labeled large neurons with a clear profile were counted. The same software was applied for determining the diameters of the labeled cells and for taking photomicrographs of selected DAB stained sections. Cells were counted in every second section across the entire PrCnF). The sections from dextran injected mouse brains and spinal cords were examined and photographed under the same microscope, the Nikon Eclipse 80i. The spinal cord sections were mapped onto diagrams extracted from the mouse spinal cord atlas of Watson et al. (2009), with reference to adjacent Nissl stained sections.

Results Retrograde labeling After cervical injections, small clusters of neurons were labeled in the PrCnF (Fig. 1a, b). In the rostralmost part of the PrCnF, there were 17.6 ± 1.6 labeled neurons per section (based on 6 sections prepared from 3 mice), situated in the proximity of the lateral periaqueductal gray

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(LPAG). In these sections, a few large, labeled neurons were intermingled with other labeled, oval-shaped cells of medium size (Fig. 1b). The average diameter of all labeled neurons in the PrCnF was 21.16 ± 1.67 lm 9 13.89 ± 1.22 lm (mean ± SD). The number of labeled neurons tapered in more caudal sections. At the level of the rostral trochlear nucleus, their number dropped to 9.8 ± 0.8 per section, but large labeled neurons (24.12 ± 0.84 lm 9 14.80 ± 0.64 lm) could still be seen. At the level caudal to the trochlear nucleus, 1–4 labeled neurons per section were observed at the border of the LPAG and the PrCnF. After counting every second section, there were 73.0 ± 9.2 and 24.3 ± 2.6 labeled cells in total on the ipsi- and contralateral sides, respectively (on the basis of 24 sections prepared from 3 mice). After upper thoracic injections, there were 15.3 ± 1.5 and 6.7 ± 2.1 labeled cells in total on the ipsi- and contralateral side of the PrCnF, respectively (on the basis of 24 sections from 3 animals; Fig. 1c, d). In terms of their size (20.07 ± 5.14 lm 9 13.29 ± 3.37 lm) and shape, the labeled neurons situated in the PrCnF were similar to those seen after cervical injections (Fig. 1d). After upper lumbar injections, labeled cells were not found in the PrCnF, but were still seen in the LPAG adjacent to the PrCnF (data not shown). Anterograde labeling In our injections, we commonly observed a small amount of spread of BDA to adjacent cell groups (PAG, SC, and mRt), but we found that the hallmark of PrCnF injections was the presence of extensive ipsilateral projections to the spinal cord. The following result was from one typical injection in the PrCnF with some spread to the adjacent SC. The injection site extended from the rostral pole of the PrCnF to a level of 320 lm caudal to the rostral pole. It involved the ventralmost part of the SC dorsally and the dorsalmost part of the mRt ventrally (Fig. 2a-a0 ). Labeled fibers mainly traveled along the ipsilateral ventral and lateral funiculi and entered the ipsilateral spinal cord from the ventral horn (Fig. 2a-a00 ). In upper cervical segments, labeled fibers entered the spinal cord from the ventral and lateral funiculi through the lateral and medial portions of lamina 8. They traveled dorsomedially towards the central canal and were distributed in the ventral portion of lamina 7, the dorsal and central portions of lamina 8, the medial portion of lamina 9, and the ventral portion of area 10 (Fig. 2b). In mid-cervical segments, labeled fibers were distributed more medially. Most of the fibers were located in the medial and central portions of lamina 8, and the ventral portion of lamina 7. Labeled fibers were also seen in the medial part of lamina 9, where the motor neurons of axial muscles were located, and the lateral part of area 10


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Fig. 1 Labeled cells in the precuneiform nucleus after spinal cord injections. a The spread of fluoro-gold in the right half of the upper cervical cord. b Labeled cells in the precuneiform nucleus after upper cervical injections. These labeled cells are located in the medial part of this nucleus, close to labeled neurons in the lateral periaqueductal gray medially. The majority of labeled neurons in the precuneiform nucleus are on the ipsilateral side. c The spread of fluoro-gold in the right half of the upper thoracic cord. d Labeled cells in the precuneiform nucleus after upper thoracic injections. Fewer labeled cells are seen in the medial part of this nucleus (note weakly labeled

cells in the lateral part). Labeled neurons can also be seen in the adjacent mesencephalic reticular formation, lateral periaqueductal gray (with an ipsilateral predominance), contralateral oral part of the pontine reticular nucleus, paralemniscal nucleus, and bilateral superior colliculus (with a contralateral predominance). The photomicrographs in b and d demonstrate the labeled neurons in the precuneiform nucleus in the rectangular area marked on the diagram. The scale bar in the photomicrograph is 50 lm. The scale bar for the injection site is 100 lm. The scale bar for the drawings is 1 mm

(Fig. 2d). As labeled fibers traveled down to the 5th and 6th cervical segments, they were more confined to the medial ventral horn. Most of them entered the spinal cord from the ventral funiculus, passing by the medial part of lamina 9, and were distributed mainly in lamina 8 and the adjacent part of medial lamina 7 (Fig. 2e). In caudal cervical segments, the density of labeled fibers dropped significantly and they were mainly distributed in lamina 8,

parallel with its long axis. To a lesser extent, fibers were also found in the ventromedial portion of lamina 7 and 9 (Fig. 2f). In upper thoracic segments, labeled fibers were mainly found in lamina 7 and to a lesser extent in the medial portion of lamina 8 (Fig. 2g). Labeled fibers were not found in lower thoracic and lumbar segments. Injections of BDA restricted to the SC resulted in contralaterally labeled fibers in the upper cervical cord only.

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Fig. 2 Labeled fibers in the spinal cord after a biotinylated dextran injection in the precuneiform nucleus. a-a0 The injections site to the PrCnF and SC (white dashed line). a-a00 Fibers from the PrCnF traveling in the ventral and lateral funiculi (arrows) at C3 level. b Fiber distribution in the 2nd cervical cord after the precuneiform nucleus injection. Labeled fibers are found in laminae 7 and 8, medial lamina 9, and area 10. c The distribution of tectospinal fibers in the contralateral C3. Labeled fibers are found in laminae 5 (arrows), 7, 8, and 9. d Fiber distribution in the 4th cervical cord after the precuneiform nucleus injection. Labeled fibers are found in lamina 7 (arrow), 8 (arrows), and to a lesser extent in lamina 9. e Fiber distribution in the 6th cervical cord after the precuneiform nucleus injection. Labeled fibers are seen in ventral lamina 7, medial laminae

8, and medial lamina 9. f Fiber distribution in the 8th cervical cord after the precuneiform nucleus injection. Labeled fibers are seen in ventral lamina 7, medial 8, and medial lamina 9. g Fiber distribution in the 2nd thoracic cord after the precuneiform nucleus injection. Labeled fibers are seen in lamina 7, to a lesser extent in lamina 8 and 9. h Fiber distribution in the 5th lumbar cord after the periaqueductal gray injection. Dense fibers are seen in lamina 5 (arrows) and dorsal lamina 6 (arrow), extending towards the central canal. The photomicrograph on the bottom right shows the periaqueductal gray injection site. The scale bar for photomicrographs and a-a00 is 50 lm. The scale bar for plates is 100 lm. The scale bar for the precuneiform nucleus injection site is 200 lm and the periaqueductal gray injection site is 500 lm

They were similarly distributed in the same spinal cord laminae as those from the PrCnF, except that some fibers from the SC were also present in lamina 5 (Fig. 2c). When BDA was injected to the adjacent PAG, especially the lateral part (LPAG), labeled fibers were most numerous in upper cervical spinal cord. In the lower cervical spinal cord, labeled fibers were distributed in the medial ventral horn similar to those from the PrCnF, but fibers in the area around central canal were denser. Labeled fibers were observed in the thoracic and lumbar spinal cords bilaterally with an ipsilateral predominance. Densely packed fibers

could be seen in lamina 5 (Fig. 2h). To a lesser extent, fibers were seen in area 10, and laminae 7 and 8.

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Discussion We have shown that the neurons of the mouse PrCnF project to the medial portions of laminae 7, 8, 9 and area 10 of the cervical and upper thoracic spinal cord. These projections are chiefly ipsilateral. The PrCnF is a major component of the presumptive MLR, so this projection


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In presenting our results, we considered the possibility that injections in the PrCnF might have labeled fibers arising in neighbouring areas, particularly the SC and the PAG. To solve this problem we studied the spinal projections from each of these areas. Our injections of retrograde tracer showed that the SC projects almost exclusively to the contralateral spinal cord, whereas the projections from the PrCnF and the PAG are mainly ipsilateral. We have concluded that the ipsilateral projections demonstrated by injection of anterograde tracer into the PrCnF could not have arisen in the SC. Moreover, the contralateral fibers labeled in the SC experiments had a different pattern of termination from those endings in the ipsilateral spinal cord. Since contralateral fibers could not be seen in segments caudal to the mid-cervical spinal cord on the contralateral side, we believe that the contralateral terminals are those arising from the SC. The projections from the PAG were ipsilateral (as were those of the PrCnF), but the pattern of termination was very different from those arising in the PrCnF. Anterograde tracer injections restricted to the PAG produced a dense pattern of labeled fibers in lamina 5 and this pattern extended to lumbar segments. Such a projection was not seen after the PrCnF injections.

projecting to the spinal cord of the mouse. However, Satoda et al. (2002) found that neurons in the area they identified as the CnF were labeled after injecting wheat germ agglutinin-horseradish peroxidase (WGA-HRP) into the central and deep regions of the ventral horn. Once again, the difference between this and our study may be based on different interpretation of the anatomy of these midbrain nuclei. Our study showed that the PrCnF fibers terminated in the medial part of lamina 7, as well as some in laminae 8 and 9, and area 10. Larger injections involving the LPAG resulted in labeled fibers in lamina 5. As noted above, the PAG projects strongly to ipsilateral lamina 5, especially in the thoracic and lower lumbar spinal cord. The laterocaudal SC projects to the contralateral laminae 5, 7, and 8, with relatively sparse distribution in lamina 9 (Yasui et al. 1998). In addition, a study on the cat revealed ipsilateral terminals originating from the VLPAG in laminae 7 and 8 (Mouton and Holstege 1994). However, the injection core also spread to the adjacent nucleus, which might have corresponded to the mouse PrCnF. In a different study on the cat and monkey, lamina 1 neurons were found to project to the caudal part of the CnF after injecting Phaseolus vulgaris leucoagglutinin (PHA-L) into lamina 1 or 1–3 (Craig 1995). It is important to note that no retrogradely labeled neurons were found in lamina 1–3 in our study after injections of dextran into the PrCnF. This discrepancy might be explained by the more caudal location of the CnF in the mouse.

Possible confusion regarding the identity of CnF and PrCnF in different species

Functional significance of the PrCnF projections to the spinal cord

There is a marked discrepancy between our findings on the spinal projection of the CnF in the mouse and the results of studies in the cat and the monkey. We found few labeled cells in the CnF after spinal injections of retrograde tracer (Liang et al. 2011), whereas Castiglioni et al. (1978) showed that the ventral portion of the monkey CnF has a substantial projection to the ipsilateral spinal cord, and Satoda et al. (2002) found that the CnF projects to the first cervical cord segment in the cat. It is possible that the neurons that we have identified in the medial portion of the PrCnF might be homologous with the medial portion of the CnF in the monkey and cat. Alternatively, the area recognised in the cat and monkey as the CnF might actually be an area that we would classify as PrCnF.

In our study, the PrCnF has been found to project ipsilaterally to the medial ventral horn of the cervical and upper thoracic spinal cord, suggesting that the mouse PrCnF might play a role as important in locomotion as its homologous region of the CnF in other mammals. In addition, the mRt, which is rostral to the PrCnF, has been found to have similar projections to the spinal cord. Therefore, it seems appropriate to propose that the MLR includes not only the mRt, but the PrCnF as well. The fact that the PrCnF projects only to cervical segments does not indicate a limited role in locomotion, since intrinsic spinal pathways (propriospinal pathways) mediate coordinated movements of the forelimb and hindlimb (Miller et al. 1998; Juvin et al. 2005; Cowley et al. 2010). In the present study, we have focused on the direct connections between the PrCnF and the spinal cord, but it is also possible that initiation of locomotion might be mediated via projections to nuclei in the hindbrain, such as the gigantocellular reticular formation (Garcia-Rill and Skinner 1987; Degtyarenko et al. 1998; Noga et al. 2003).

might be a pathway by which the MLR initiates locomotor activity. Potential overlap between projections from the PrCnF and those of the PAG and the SC

The pattern of termination of PrCnF fibers in the spinal cord No detailed studies have been hitherto published describing the terminal pattern of fibers originating from the MLR and

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Most of the terminals from the PrCnF ended in laminae 7 and 8, and only a few ended among the motor neurons in lamina 9. We conclude that most of the spinal motor neurons stimulated by PrCnF axons must be activated via interneurons. It is well known that there is a central pattern generator for locomotion in the spinal cord, which is located in laminae 7, 8, and area 10 (Bracci et al. 1996; Cina and Hochman 2000; Dai et al. 2005; Kjaerulff et al. 1994; Kjaerulff and Kiehn 1996; Tresch and Kiehn 1999). The interneurons (in lamina 7 and 8) of the central pattern generator, which are rostral to the motor neurons that directly innervate the limb muscles, also produce rhythmic activity in the limbs (Cazalets et al. 1995; Bertrand and Cazalets 2002) and there is a capacity gradient along the rostrocaudal axis of the spinal cord (Grillner and Zangger 1979; Bracci et al. 1996; Bonnot and Morin 1998; Bonnot et al. 2002; Christie and Whelan 2005; Magnuson et al. 2005). Hence, there exists a possible anatomical substrate for the role of PrCnF projections to the spinal cord in the initiation of locomotion. It is interesting to note that spinal cord neurons in lamina 1 send afferents to the CnF (Bjo¨rkeland and Boivie 1984; Hylden et al. 1989). Therefore, it seems reasonable to suggest that these ascending projections might form the afferent pathway of a nociceptive reflex circuit, which utilizes descending fibers originating from the PrCnF to target spinal motor neurons or interneurons to avoid noxious stimuli. Acknowledgments We thank Dr Yuhong Fu, Dr Yue Qi, Dr Erika Gyengesi and Mr Peter Zhao for their suggestions and technical support, and Dr Zoltan Rusznak for the manuscript corrections. This work was supported by The Christopher & Dana Reeve foundation and an NHMRC Australia Fellowship grant to Professor George Paxinos (466028).

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