THE JOURNAL OF COMPARATIVE NEUROLOGY 461:429 – 440 (2003)
Synaptic Targets of Commissural Interneurons in the Lumbar Spinal Cord of Neonatal Rats ´ S BIRINYI,1 KORNE ´ L VISZOKAY,1 ILDIKO ´ WE ´ BER,1 OLE KIEHN,2 ANDRA ´ S ANTAL1* AND MIKLO 1 Department of Anatomy, Histology and Embryology, Faculty of Medicine, Medical and Health Science Center, University of Debrecen, Debrecen, H-4012 Hungary 2 Mammalian Locomotor Laboratory, Department of Neuroscience, Karolinska Institutet, Stockholm, S-17777 Sweden
ABSTRACT There is strong evidence that commissural interneurons, neurons with axons that extend to the contralateral side of the spinal cord, play an important role in the coordination of left/right alternation during locomotion. In this study we investigated the projections of commissural interneurons to motor neurons and other commissural interneurons on the other side of the spinal cord in neonatal rats. To establish whether there are direct contacts between axons of commissural interneurons and motor neurons, we carried out two series of experiments. In the first experiment we injected biotinylated dextran amine (BDA) into the lateral motor column to retrogradely label commissural interneurons that may have direct projections to motor neurons. Stained neurons were recovered in the ventromedial areas of the contralateral gray matter in substantial numbers. In the second experiment BDA was injected into the ventromedial gray matter on one side of the lumbar spinal cord, whereas motor neurons were simultaneously labeled on the opposite side by applying biocytin onto the ventral roots. BDA injections into the ventromedial gray matter labeled a strong axon bundle that arose from the site of injection, crossed the midline in the ventral commissure, and extensively arborized in the contralateral ventral gray matter. Many of these axons made close appositions with dendrites and somata of motor neurons and also with commissural interneurons retrogradely labeled with BDA. The results suggest that commissural interneurons may establish monosynaptic contacts with motor neurons on the opposite side of the spinal cord. Our findings also indicate that direct reciprocal connections between commissural interneurons on the two sides of the spinal cord may also exist. J. Comp. Neurol. 461:429 – 440, 2003. © 2003 Wiley-Liss, Inc. Indexing terms: motor neurons; locomotion; neural tracing; biotinylated dextran amine; biocytin
The spinal cord plays a central role in generating rhythmic movements like walking, running, and scratching. Intrinsic spinal networks control much of motor neuron activities that underlie these rhythmic motor behaviors. The spinal rhythm-generating networks that provide the phasic activation of motor neurons during rhythmic movement (Grillner et al., 1995; Kiehn et al., 1997) are found in all vertebrates, including man, and are known as central pattern generators (CPGs). Neurons that coordinate activities between the left and right sides of the spinal cord constitute essential elements of the CPG. Many of these neurons, regarded as commissural interneurons (CINs), possess axons that cross the midline and project to the opposite side of the ventral gray matter. CINs have been the subject of a series of studies conducted recently in our laboratories. Using the isolated neonatal rat spinal cord that produces long-lasting alternating rhythmic activity © 2003 WILEY-LISS, INC.
in the presence of various neuroactive substances (Kiehn and Kjaerulff 1998; Schmidt et al., 1998; Cazalets et al.,
Grant sponsor: the Hungarian National Research fund; Grant number: OTKA 032075; Grant number: OTKA 037522; Grant sponsor: Scientific Council of the Ministry of Welfare, Hungary; Grant number: ETT 04-32/ 2000 (MA and BA); Grant sponsor: National Institutes of Health; Grant sponsor: the Karolinska Institute, Stockholm, Sweden (OK). *Correspondence to: Miklo´s Antal, Department of Anatomy, Histology and Embryology, Faculty of Medicine, Medical and Health Science Center, University of Debrecen, Debrecen, H-4012 Hungary. E-mail:
[email protected] Received 4 September 2002; Revised 9 December 2002; Accepted 17 January 2003 DOI 10.1002/cne.10696 Published online the week of May 19, 2003 in Wiley InterScience (www. interscience.wiley.com).
430 1998), we focused our attention on the anatomical and physiological characterization of CINs (Kjaerulff and Kiehn 1996, 1997; Eide et al., 1999; Stokke et al., 2002; Butt et al., 2002). We demonstrated that neurons that are involved in the generation of left/right alternation during fictive locomotion are located in the ventromedial area (medial part of laminae VII, X, and VIII) of the lumbar spinal cord in neonatal rats (Kjaerulff and Kiehn, 1996). Tracing studies have also shown that this area contains four major groups of CINs with different axonal projections: long-range (more than 1.5 segment) projecting CINs with ascending, descending, or bifurcating axons, and short-range (less than 1.5 segment) projecting CINs (Stokke et al., 2002). In lampreys and tadpoles it has been demonstrated that at least some CINs establish direct contacts with motor neurons and other CINs (Grillner et al., 1995; Roberts et al., 1998), and thus may play a crucial role in the coordination of motor activities on the two sides of the spinal cord. However, in mammals there is only indirect evidence for direct projections of CINs to contralateral motor neurons (Kjaerulff and Kiehn 1997; Jankowska and Skoog, 1986) and there is no account in the literature on reciprocal CIN-CIN connections. Thus, using retrograde and anterograde tracing techniques, the aim of the present study was to investigate whether CINs located in the ventromedial gray matter make direct contacts with motor neurons and/or other CINs in the contralateral side of the spinal cord of neonatal rats. Preliminary observations from this experiment have been published previously in abstract form (Birinyi et al., 2001).
MATERIALS AND METHODS Dissection, labeling, and preparation of tissue sections Experiments were performed on the isolated spinal cords of 23 neonatal (1– 4 days old) Wistar rats. The animal study protocols were approved by ethical committees at both the University of Debrecen, Hungary, and Karolinska Institute, Stockholm, Sweden, and were in accordance with NIH guidelines. As described previously (Kiehn at al., 1996), the animals were decapitated and eviscerated under deep ether anesthesia. The spinal cord was exposed by a ventral laminectomy and carefully dissected sparing the ventral and dorsal roots. The dissected cord was transferred to a recording chamber superfused with Ringer’s solution (pH 7.4) saturated with 5% CO2 in O2 and contained (in mM): NaCl (128), KCl (4.69), NaHCO (25), KH2PO4 (1.18), MgSO4 (1.25), CaCl2 (4.52), and glucose (22). Glass micropipettes with a tip diameter of 15–20 m were filled with a 10% solution of biotinylated dextran amine (BDA, molecular weight 10,000; Molecular Probes, Eugene, OR, USA) dissolved in 0.1 M phosphate buffer (PB, pH 7.4). The tracer was injected unilaterally into the lateral motor column or into the ventromedial area of the gray matter at the level of the L2 and L4 spinal segments of the lumbar spinal cord by using positive direct current with an amplitude of 5–15 A, duty cycle of 50% and injection period of 5 minutes. Following BDA injections into the ventromedial gray matter, motor neurons contralateral to the injection were also labeled by placing the L2–L5 ventral roots on a small piece of parafilm floating on the surface of the Ringer’s solution and applying bio-
A. BIRINYI ET AL. cytin crystals (Sigma, St. Louis, MO, USA) to the stumps for 20 – 40 minutes. After tracer application the spinal cords were kept in the recording chamber for 4 – 8 hours and then transferred into a fixative containing 2.5% glutaraldehyde, 0.5% paraformaldehyde, and 0.2% picric acid in 0.1 M PB. The lumbar segments of the spinal cord were sectioned at 60 m on a vibratome and washed extensively in 0.1 M PB.
Histochemical procedure For histochemical detection of BDA and biocytine, freefloating sections were incubated with avidin-biotinylated peroxidase complex (ABC, 1:100 Vector Laboratories, Burlingame, CA) overnight at 4°C. Sections were extensively washed in 0.1 M PB and 0.05 M TRIS and the histochemical reaction was completed with a nickel-intensified diaminobenzidine chromogen reaction (Hancock, 1984). Finally, the sections were washed in 0.05 M TRIS and 0.1 M PB, mounted on gelatin-coated slides, and coverslipped with Permount neutral medium (Fischer Scientific, Pittsburgh, PA, USA).
Reconstruction of labeled neurons The distribution and morphology of retrogradely labeled spinal interneurons were investigated in serial sections of the spinal cord. The dendritic trees of labeled commissural interneurons were reconstructed using a camera lucida. Camera lucida drawings were made in such a way that the neurons located in consecutive sections were summarized in one drawing. Labeled neurons were photographed using a cooled SPOT RT CCD camera (Diagnostic Instruments, Burroughs, MI, USA). The brightness and contrast of the images were adjusted using the Adobe PhotoShop software package (Adobe Systems, Mountain View, CA, USA). Presumed contacts between CINs and motor neurons were identified with the use of a 100⫻ oil-immersion objective. Appositions were regarded as contacts if no gap was discerned between labeled axon varicosities of CINs and somata or dendrites of motor neurons.
RESULTS On the basis of the location of BDA injections and the application of biocytin to the ventral roots, the animals were divided into two experimental groups. In some animals BDA was injected into the lateral motor column at the level of L2 and L4 spinal segments (first experimental group). In other animals BDA was delivered into the ventromedial gray matter at the level of L4 spinal segments and biocytin was simultaneously applied to the L2–5 ventral roots contralateral to the site of BDA injections (second experimental group). The experiments were carried out on 23 animals. Because of inappropriate localization of the injection sites and insufficient labeling of motor neurons, 13 animals had to be excluded from the final evaluation. Consequently, the data presented in this article are based on the results obtained in 10 animals: three animals in the first and seven animals in the second experimental group.
Injections of BDA into the lateral motor column BDA was delivered into the lateral motor column at the level of the L2 and L4 segments of the lumbar spinal cord
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Fig. 1. Injection of BDA into the lateral motor column. A: Schematic representation of how BDA was injected into the lateral motor column. Last-order premotor interneurons (LOPI) and last-order premotor commissural interneurons (LOPCIN) that can primarily be labeled with this method are also illustrated. Shaded area represents the territory that is supposed to be infiltrated by the tracer. B: Photomicrograph showing an injection site of BDA. Dashed lines on inserts A and B represent the border between the gray and white matters. C,D: Photomicrographs illustrating retrogradely labeled interneurons contralateral to the injection site. cc, central canal. Scale bars ⫽ 100 m in B, 25 m in C and D.
(Fig. 1A). The iontophoretic injection of BDA yielded a small necrotic center surrounded by a shell of diffusely stained region (Fig. 1B). The necrotic center with the diffusely stained outer rim that was regarded as the site of injection involved a cross-sectional area of 100 –150 m in diameter and extended approximately the same length along the rostrocaudal axis of the spinal cord. Following BDA injections into the motor column, large numbers of retrogradely labeled neurons were encountered in the lumbar spinal gray matter both ipsi- and contralateral to the site of tracer application (Fig. 2). Despite the substantial ipsilateral labeling we focused our attention exclusively on neurons that were labeled in the contralateral gray matter. Most of these neurons showed a homogeneous intense staining that labeled both the somata and a large part of the dendritic trees (Fig. 1C,D). Forty-seven (42%) of the 112 investigated cells presented pyramidal or multipolar cell bodies, the diameter of which varied in the range of 10 –20 m (Fig. 1C). Sixty-five (58%) neurons had fusiform cell bodies with diameters of 20 –30 m (Fig. 1D). The perikarya gave rise to three or four stem dendrites
that arborized once or twice near the cell body. The secondary or tertiary branches extended more or less straight over a distance of several hundreds of micrometers in various directions in the spinal gray matter (Fig. 1C,D). Although most of the labeled neurons were located ipsilateral to the injection site (83% and 82% in injections at the level of the L2 and L4 spinal segments, respectively), stained neurons were recovered in the contralateral gray matter also in substantial numbers (17% and 18% in injections at L2 and L4 segments, respectively) (Fig. 2, Table 1). Ipsilateral to the site of injections, labeled neurons were mostly confined to laminae V–VIII and were distributed over the entire medio-lateral extent of these laminae (Fig. 2). In contrast to this, neurons that were recovered in the contralateral gray matter were concentrated in the ventromedial gray matter corresponding mostly to lamina VIII and partly to the medial part of lamina VII and lamina X (Fig. 2). Regarding the rostrocaudal distribution of the labeled neurons, most of them were located within the segment of the injection site (Fig. 2F,G). However, some of them were
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Fig. 2. Distribution of retrogradely labeled spinal interneurons following BDA injections into the lateral motor column at the level of L2 (A,B,D,F) and L4 (C,E,G) spinal segment. A–C: Camera lucida drawings of transverse sections of the lumbar spinal cord showing the distribution of retrogradely labeled spinal interneurons in animals BDA20, BDA19, and BDA18. The drawings illustrate all labeled cells that were recovered from serial sections at the level of L1–2, L3– 4, and L5– 6 spinal segments. Each dot represents one labeled neuron. Shaded areas represent the sites of BDA injections at the level of L2 (A: animal BDA20, B: animal BDA19) and L4 (C: animal BDA18) spinal segment. D–G: Histograms showing the laminar (D,E) and segmental (F,G) distribution of all retrogradely labeled neurons ipsi- (IPSI) and contralateral (CONTRA) to the site of BDA injection in animals BDA20 and BDA19 (D,F) as well as in animal BDA18 (E,G). Roman numerals on inserts D and E correspond to Rexed laminae of the spinal gray matter. Each bar is proportional to the percentage of cells located in the indicated laminae or segments.
scattered in more distant positions, rostral or caudal to the segment of BDA application. In the case of injections at L2, neurons were recovered in substantial numbers both rostral (in L1 segment) and caudal (in L3 and L4 segments) to the injection site (Fig. 2F). In the case of injections at L4, however, neurons were almost exclusively found rostral to the site of BDA delivery (Fig. 2G). Stained cells were encountered in L5 and L6 segments only in limited numbers in both cases (Fig. 2A–C,F,G)
TABLE 1. Number of Labeled Spinal Interneurons Following Injections of BDA into the Motor Column Labeled spinal interneurons Animal
Injection
Total
BDA 20 BDA 19 BDA 18
L2-LMC L2-LMC L4-LMC
1092 558 948
Ipsilateral 821 466 779
83% 83% 82%
Contralateral 169 92 169
17% 17% 18%
L2, L4, number of segments of the lumbar spinal cord; LMC, lateral motor column.
SYNAPTIC TARGETS OF COMMISSURAL INTERNEURONS These findings suggested that labeled neurons located in the ventromedial gray matter contralateral to the site of BDA injections may provide a substantial direct projection to motor neurons on the other side of the spinal cord. To further investigate this possibility we carried out a second set of experiments.
Injections of BDA into the ventromedial gray matter and simultaneous application of biocytin to ventral roots on the opposite side On the one hand, BDA was delivered into the ventromedial gray matter at the level of the L4 segments of the lumbar spinal cord (Fig. 3A). Similar to deliveries into the lateral motor column, the iontophoretic injection of BDA yielded a small necrotic center surrounded by a shell of diffusely stained region that involved a cross-sectional area of 50 –150 m in diameter (Fig. 3B). To label motor neurons biocytin was applied to the L3–5 ventral roots contralateral to the site of BDA injections (Fig. 3A). Application of biocytin to the ventral roots resulted in retrograde labeling of a large number of motor neurons (Fig. 3D). In addition to a strong labeling of perikarya, substantial proportions of the dendritic trees were also stained (Fig. 3D). Commissural interneuron–motor neuron connections. BDA injections into the ventromedial gray matter labeled a strong axon bundle that arose from the site of injection, crossed the midline in the ventral commissure, and extensively arborized in the contralateral ventral gray matter (Fig. 3C). Many of the axons could be followed as far as the lateral motor column, where they formed close appositions with somata and dendrites of retrogradely labeled motor neurons (Fig. 4). Looking for close appositions formed by the anterogradely labeled commissural axon terminals on retrogradely stained motor neurons, we traced the cell body and the dendritic tree of 632 motor neurons located at different segments of the spinal cord (Table 2). Careful analysis of the sections revealed close appositions between labeled axon terminals and cell bodies and/or dendrites of motor neurons in 291 cases. Individual motor neurons received 1 to 3 contacts from labeled axon terminals. Nearly one-fourth of the appositions were found on somata (Fig 4A, Table 2), whereas the others were revealed on dendrites (Fig. 4B, Table 2). The dendritic appositions were found mostly, if not exclusively, on proximal dendrites. In addition to axons of contralateral CINs, labeled terminals that establish close appositions with motor neurons may also represent terminals of initial axon collaterals of motor neurons in our double-labeling experiments. To evaluate the contribution of these initial axon collaterals to the total numbers of contacts, we traced the axons of 167 retrogradely labeled motor neurons in three preparations where only the motor neurons were labeled. We found no initial axon collaterals on the labeled motoneurons. Commissural interneuron– commissural interneuron connections. Following BDA injections into the ventromedial gray matter, in addition to the commissural axons large numbers of retrogradely labeled neurons were also recovered in both sides of the spinal gray matter (Figs. 3B,C, 5).
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Although, depending on the size and the exact location of the injection site, the numbers and distribution of the retrogradely labeled cells varied in a wide range, neurons in the ventromedial gray matter contralateral to the site of injection were always revealed in substantial numbers (Fig. 5A–E, Table 3). Most of these cells were located within the segment of the injection site (Fig. 5A–C,G). However, some of them were scattered in more distant positions, rostral or caudal to the segment of BDA application (Fig 5A–C,G). Most of these neurons presented multipolar, pyramidal, or fusiform cell bodies with diameters of 10 –30 m (Fig. 6). The perikarya gave rise to 2–5 stem dendrites that branched once or twice near the cell body and then extended more or less straight into various directions within the spinal gray matter (Fig. 6). Thorough investigation of the somata and dendritic trees of these neurons showed that many of them established close appositions with labeled axon terminals (Fig. 7). Of the 35 neurons examined this way 17 were apposed by labeled axons. Individual neurons received 1 to 4 contacts. The appositions were found exclusively on proximal dendrites (Fig. 7).
DISCUSSION It has previously been demonstrated that neurons that are involved in the generation of left/right alternation during locomotion are located in the ventromedial area of the lumbar spinal cord in neonatal rats (Kjaerulff and Kiehn, 1996). It is also established that many of these neurons in the ventromedial gray matter possess axons that cross the midline and terminate in the contralateral gray matter (Puska´ r and Antal, 1997; Eide et al., 1999; Stokke et al., 2002), and thus can be regarded as commissural interneuron (CIN). Conveying neural signals from one side of the spinal cord to the other, CINs are essential elements of neural circuits underlying left/right coordination during locomotion. Using single- and double-labeling neural tracing techniques, we demonstrate for the first time that axons of CINs in the ventromedial gray matter of the lumbar spinal cord of neonatal rats project directly to perikarya and dendrites of contralateral motor neurons. We also show that ventromedial CINs on one side of the spinal cord project to other CINs located on the opposite side of the spinal cord. These results argues that ventromedial CINs may modulate left/right coordination of motor behaviors in a complex way: partly acting directly on motor neurons, partly regulating the input– output properties of other CINs.
Labeling of neurons with biotinylated dextran amine It is well established that BDA shows excellent properties for both anterograde and retrograde neural labeling (Veenman et al., 1992; Rajakumar et al., 1993). BDA is taken up by axon terminals, dendrites, and cell bodies and is transported both anterogradely and retrogradely to yield Golgi-like labeling of axon terminals and the somatodendritic compartment of neurons. In our studies BDA was injected into the lateral motor column and the ventromedial aspect of the gray matter. In the case of lateral motor column injection, it appears likely that most of the retrogradely labeled cells within the ven-
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Fig. 3. Injection of BDA into the ventromedial gray matter and simultaneous labeling of motor neurons with biocytin on the opposite side of the spinal cord. A: Schematic representation of how BDA was injected into the ventromedial gray matter and how motor neurons were retrogradely labeled with biocytin. Last-order premotor commissural interneurons (LOPCIN), motor neurons (MN) and commissural interneurons (CIN) that can primarily be labeled with this method are also illustrated. Shaded area represents the territory that is supposed to be infiltrated by the tracer. B: Photomicrograph showing an injec-
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tion site of BDA. Dashed lines on inserts A and B represent the border between gray and white matters. C: Photomicrograph illustrating anterogradely labeled axons of commissural interneurons (arrowheads) that cross the midline in the anterior commissure. Note that injections of BDA into the ventromedial gray matter also labeled interneurons (arrows) on the opposite side of the spinal cord. D: Photomicrographs showing motor neurons retrogradely labeled with biocytin. cc, central canal. Scale bars ⫽ 100 m in B, 50 m in C,D.
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Fig. 4. Photomicrographs showing close appositions (arrows) between anterogradely labeled axon terminals of commissural interneurons and cell bodies (A) as well as proximal dendrites (B) of retrogradely labeled motor neuron. Scale bars ⫽ 10 m.
TABLE 2. Number of Contacts on Motor Neurons Number of contacts on motor neurons
Number of Investigated motor neurons
Contacted motor neurons
In total
On somata
On dendrites
2 4 6 7 11
130 173 98 120 111
55 78 58 50 50
75 98 79 69 71
20 22 18 21 9
55 52 61 48 62
TOTAL
632
291
392
90
302
BDA BDA BDA BDA BDA
tromedial aspects of the gray matter contralateral to the site of tracer injection can be interpreted as last-order premotor commissural interneuron (LOPCIN; see discussion in Puska´ r and Antal, 1997); that is, neurons with axons terminating on the somatodendritic compartment of contralateral motor neurons. However, in addition to motor neurons the lateral motor column may also contain cell bodies and dendrites of interneurons. Axon terminals that took up BDA in the motor column and transported it back to their perikarya might also contact these interneurons. Thus, there might be neurons among the retrogradely labeled cells that cannot be regarded as LOPCIN. Although the labeling of non-LOPCINs following BDA injection into the lateral motor column cannot be excluded, it is likely that non-LOPCINs represent a minority of the total number of retrogradely labeled cells. It has been demonstrated in the chick spinal cord that interneurons are scattered among the motor neurons in minimal numbers (Antal and Polga´ r, 1993; Antal et al., 1994; Berki et al., 1995). Moreover, dendrites of interneurons located in the ventral gray matter extend into various directions, but they hardly ever enter the lateral motor column (Antal and Polga´ r, 1993). Therefore, it seems likely that in the case of lateral motor column injections the majority of axon terminals of the retrogradely labeled neurons establish contacts with motor neurons in the lateral motor column, and thus most of the labeled cells can be considered LOPCINs. In principle, in addition to axon terminals of LOPCINs, fibers of passage crossing the injection site could also be
labeled and, consequently, cells of origin of long propriospinal and supraspinally ascending fibers might be among the stained cells in the contralateral ventromedial gray matter. It is likely, however, that this mechanism did not play a significant role in our experiment. Although it is generally assumed that BDA is taken up by fibers of passage (Veenman et al., 1992; Rajakumar et al., 1993; Dolleman-Van der Weel et al., 1994; Sidibe´ and Smith, 1996), it has also been demonstrated that the tracer can be transported only by damaged fibers of passage and not by intact fibers (Veenman et al., 1992; Rajakumar et al., 1993; Sidibe´ and Smith, 1996). Several observations indicate that, if the tracer is delivered with iontophoresis, as has been done in our experiments, the injection causes minimal tissue damage, and the uptake and subsequent transport of BDA by fibers of passage is negligible (Rajakumar et al., 1993; Sidibe´ and Smith, 1996; Puska´ r and Antal, 1997) Consequently, although we cannot rule out the possibility of minimal labeling of fibers of passage, it seems likely that this nonspecific labeling was insignificant. In the case of BDA injections into the ventromedial gray matter, the principal emphasis was put onto the recovery of labeled axon terminals and perikarya on the opposite side of the spinal gray matter. Labeling of fibers of passage might cause false staining of cell bodies and axon terminals here, too. However, for similar reasons, as in the case of lateral motor column injections, it is very likely that fibers of passage were labeled only in limited numbers in this case as well. When BDA was injected into the ventromedial gray matter on one side of the lumbar spinal cord, motor neurons were simultaneously labeled on the opposite side by applying biocytin onto the ventral roots to study the projections of CINs to contralateral motor neurons. To identify the contacts between axon terminals of CINs and motor neurons, we were looking for labeled axon terminals that established close appositions with somata and dendrites of retrogradely labeled motor neurons. However, in principle, the labeled axon terminals might arise from two sources in this experiment. They might really represent axon terminals of contralateral CINs, but they might also
Fig. 5. Distribution of retrogradely labeled spinal interneurons following BDA injections into the ventromedial gray matter at the level of L4 spinal segment. A–C: Camera lucida drawings of transverse sections of the lumbar spinal cord showing the distribution of retrogradely labeled spinal interneurons in animals BDA11, BDA14, and BDA15. The drawings illustrate all labeled cells that were recovered from serial sections at the level of L1–2, L3– 4, and L5– 6 spinal
segments. Each dot represents one labeled neuron. Shaded areas represent the sites of BDA injections. D–G: Histograms showing the laminar (D,E) and segmental (F,G) distribution of all retrogradely labeled neurons ipsi- (D,F) and contralateral (E,G) to the site of BDA injection. Roman numerals on inserts D and E correspond to Rexed laminae of the spinal gray matter. Each bar is proportional to the percentage of cells located in the indicated laminae or segments.
SYNAPTIC TARGETS OF COMMISSURAL INTERNEURONS belong to initial axon collaterals of motor neurons that were retrogradely labeled with biocytin. Accordingly, we have to raise the question of which magnitude the possible labeling of initial axon collaterals of motor neurons could bias our results. It seems that only in a minimal, if any, extent. That is, our results show that motoneurons in the neonatal rat lumbar spinal cord do not possess initial axon collaterals. Moreover, using differential fluorescent labeling of CIN axons and motor neurons with rhodaminedextran and fluorescene-dextran, respectively, close appositions between axon terminals of CINs and motor neurons were revealed in substantial numbers, but initial axon collaterals of motor neurons were not detected (unpubl. obs.).
Distribution and morphology of last-order premotor commissural interneurons Laminar distribution. The ventromedial area of the spinal gray matter has long been known to contain neurons with axons that cross to the other side of the spinal cord (Ramon y Cajal, 1909; Scheibel and Scheibel, 1969; Matsushita, 1970). Although some of these commissural neurons turned out to give rise to various supraspinally
TABLE 3. Number of Labeled Spinal Interneurons following Injection of BDA into the Ventromedial Gray Matter Labeled spinal interneurons Animal
Injection
Total
BDA 11 BDA 14 BDA 15
L4-VM L4-VM L4-VM
807 406 148
Ipsilateral 538 330 102
67% 81% 69%
Contralateral 269 76 46
33% 19% 31%
L4, number of segment of the lumbar spinal cord; VM, ventromedial gray matter.
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ascending fibers, many of them have been identified as interneurons with axons that terminate in the contralateral spinal gray matter (Szenta´ gothai, 1951; Willis and Willis, 1966; Grillner and Hongo, 1972; Yoshida et al., 1998; Eide et al., 1999; Stokke et al., 2002). Some of these interneurons have been successfully labeled by injecting the retrograde transneural tracer wheat germ agglutinin into muscles of the lower limb in cats (Harrison et al., 1985; Jankowska and Skoog, 1986) and by injecting biotinylated dextran amine into the motor column of the rat lumbar spinal cord (Puska´ r and Antal, 1997), indicating that a proportion of commissural interneurons in the ventromedial aspect of the spinal gray matter may project directly to contralateral motor neurons. Our present findings are consistent with these previous observations. Injecting BDA into the motor column at the level of L2 and L4 segments of the neonatal rat spinal cord, retrogradely labeled neurons contralateral to the site of tracer application were confined, with a few exceptions, to the ventromedial aspect of the gray matter: mostly to lamina VIII and partly to lamina X and the ventromedial part of lamina VII. In addition to these findings we also presented morphological evidence that axon terminals of commissural interneurons in the ventromedial gray matter establish close appositions with somata and proximal dendrites of motor neurons. Thus, at least a proportion of commissural neurons in the ventromedial gray matter in neonatal rats seems to be last-order premotor interneurons (LOPCIN). Since we labeled commissural interneurons almost exclusively in the ventromedial gray matter, we propose that LOPCINs are mainly confined to this area. In order to make this conclusion even stronger, ultrastructural studies are needed to verify the close appositions
Fig. 6. Photomicrographs of retrogradely labeled commissural interneurons following injection of BDA into the ventromedial gray matter. CC, central canal. Scale bars ⫽ 25 m.
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Fig. 7. Photomicrographs (A) and camera lucida drawings (B,C) illustrating close appositions between anterogradely labeled axon terminals the cell body of which was located within the confines of ventromedial BDA injections and dendrites of commissural interneurons that were labeled on the opposite side of the spinal cord by the
same BDA injection. Arrowheads in A and B indicate the same contact. Sites of close appositions are marked by black dots on B and C. Rectangle in B indicates the medial (M) and ventral (V) directions. Scale bars ⫽ 10 m in A, 50 m in B,C.
between axon terminals of potential LOPCINs and contralateral motor neurons as real synaptic contacts. Segmental distribution. Injecting WGA-HRP into peripheral nerves of the hindlimb, Harrison et al. (1985) recovered transneuronally labeled neurons in lamina VIII contralateral to the tracer injection from L4 to S1 segments in the cat. Puska´ r and Antal (1997) reported that injecting BDA into the motor column at the level of L1–L2 and L4 –L5 segments of the adult rat spinal cord LOPCINs were encountered in a three-to-four segment-long compartment of the lumbar cord. Here we demonstrated that the rostrocaudal distribution of LOPCINs in the neonatal rat is wider than that in adult animals. After injecting BDA into a 100 –150 m-long section of the motor column either at the level of L2 or L4 segments, LOPCINs were found throughout the entire length of the lumbar spinal cord. Thus, motor neurons in a 100 –150 m-long section of the motor column may receive a convergent input from LOPCINs distributed throughout the entire length of the lumbar spinal cord. However, most of the LOPCINs are located at or very close to the segmental level of the innervated motor neurons, although some may establish positions three to four segments away and innervate the contralateral motor neurons with long ascending and descending axons. This notion is strongly reinforced by the findings that the ventromedial gray matter of the neonatal rat contains CINs that possess ascending and/or descending axons that extend as long as four to five spinal segments (Eide et al., 1999; Stokke et al., 2002). Although the segmental distribution of LOPCINs is wide in the neonatal rat, it appears to be uneven along the rostrocaudal axis of the lumbar spinal cord. After injecting BDA into the motor column either at L2 or L4, LOPCINs were labeled in large numbers at the level of L1–L4, but were recovered only in limited numbers at L5–L6. This observation indicates that while L1–L4 segments contain a substantial number of LOPCINs with ascending and/or
descending axons (Eide et al., 1999; Stokke et al., 2002), L5–L6 segments contain only a few LOPCINs with ascending axons. However, following BDA injections into the ventromedial gray matter at L4, in addition to neurons at L1–L4, a substantial number of stained cells was found also in the ventromedial gray matter of L5–L6 segments. These findings suggest that the ventromedial gray matter at L5–L6 also contains CINs with ascending axons in considerable numbers, but these ascending axons terminate primarily in the contralateral ventromedial gray matter with few monosynaptic projections to contralateral motor neurons. Puska´ r and Antal (1997) also demonstrated that in the adult rat, LOPCINs were labeled in substantial numbers in animals in which BDA was injected into the medial motor column. However, after lateral motor column injections LOPCINs were recovered only in limited numbers; they represented no more that 2– 6% of the total population of the retrogradely labeled neurons. Data presented here do not appear to be in agreement with the findings of Puska´ r and Antal (1997). After delivering BDA at the level of L4, where only the lateral motor column exists and there is no representation of the medial motor column, we found as many LOPCINs in the neonatal rat as after injections at L2, where the medial motor column is well developed (18% and 17% of the total number of labeled neurons, respectively). This clear difference between the adult and neonatal animals indicates that LOPCINs projecting to motor neurons in the lateral motor column may undergo a substantial postnatal developmental reorganization. Some of the LOPCINs projecting to the lateral motor column that exist in the neonate might be lost during postnatal development due to postnatal programmed cell death. Alternatively, some LOPCINs may change their axonal arborization during postnatal development so that their monosynaptic connections to motor neurons will be lost, and only di- or polysynaptical CIN-
SYNAPTIC TARGETS OF COMMISSURAL INTERNEURONS motoneuron pathways will be left intact (Jankowska and Noga, 1990). Such connections are known to exist segmentally in the neonatal rat (Kjaerulff and Kiehn, 1997). During locomotion, corresponding muscles in the two limbs (e.g., hip flexors) must contract in precise alternation, while flexors and extensors in opposite limbs often contract in synchrony. Part of this coordination could be mediated through direct LOPCIN-motor neuron connections both at the segmental and intersegmental level. Thus, segmental LOPCIN-motor neuron connections could serve a critical role in the strict alternation observed segmentally, while LOPCINs with long ascending and descending axons may be involved in a longitudinal coupling system that ensures a stable multijoint coordination during locomotion. Somatodendritic morphology. This is the first account in which the somatodendritic morphology of LOPCINs was studied in a relatively large sample. We have reported that, despite the fact that labeled neurons showed a high degree of variability in their morphology, on the basis of the size and shape of the cell bodies and the dendritic arborization patterns, the neurons could be divided into two morphological categories: 1) neurons with pyramidal or multipolar perikarya, and 2) fusiform cells. The classification of LOPCINs based on these simple morphological criteria may be helpful in further characterization of this neuronal population.
Reciprocal connections between commissural interneurons in the ventromedial gray matter Several previous studies have defined the ventromedial gray matter of the mammalian spinal cord as important for the generation of locomotion (Kremer and Lev-Tov, 1997; Tresch and Kiehn, 1999). This area has also been shown to contain neuronal elements, including CINs that are necessary for left/right coordination of limb movements (for review, see Kiehn and Kjaerulff, 1998). A number of physiological studies carried out on aquatic vertebrates have shown that ventromedial CINs on the two sides of the spinal cord form reciprocal interactions with each other (Buchanan, 1982, 1996; Dale 1985; Roberts, 1990). This reciprocal CIN–CIN interactive system has been shown to be a key element in the coordination of swimming behavior (Grillner et al., 1995; Arshawsky et al., 1993). With the application of morphological methods, here we show for the first time that a similar arrangement appears to be present also in the mammalian spinal cord. This conclusion is based on the interpretation of the finding that CINs receive close appositions from axon terminals that are labeled with injections of BDA into the opposite ventromedial gray matter. However, in addition to contralateral CINs these axon terminals may also arise from retrogradely labeled recurrent axon collaterals of ipsilateral CINs. Although in principle this possibility cannot be ruled out, it is very likely that retrograde labeling of recurrent axon collaterals is minimal in our samples. That is, previous studies indicate that CINs may possess ipsilateral recurrent axon collaterals very rarely. Labeling of CINs by injections of fluorescent tracers into various areas of the ventral spinal gray matter in the rat lumbar spinal cord, initial axon collaterals that arborize and terminate ipsilateral to the cells of origin were encountered only in limited, if any, numbers (Eide et al.,
439
1999). Thus, it is highly conceivable that most of the contacts between labeled axon terminals and CINs contralateral to the ventromedial BDA injections represent commissural CIN–CIN interactions. The finding that CINs contacted by commissural axons were recovered not only at the segmental level of tracer application, but also in adjacent segments both rostral and caudal to the site of BDA injections, indicates that CINs may form monosynaptic contacts with each other not only at the segmental level but intersegmental direct CIN–CIN interactions may also exist. The intrasegmental and intersegmental CIN–CIN connections may serve different roles in binding muscles into alternation or synchrony during locomotion. Our results also suggest that these reciprocal interactions may be powerful, since a substantial proportion of the investigated CINs was found to be apposed by axon terminals of contralateral CINs. Whether axon collaterals of LOPCINs or axons of other CINs, or both, are involved in the formation of this reciprocal interactive system cannot be determined from our present set of data. Future studies aimed at characterizing the transmitter content of CINs and LOPCINs is an obvious next step in revealing the functional role of this anatomically defined reciprocal CIN–CIN interactive system of the mammalian spinal cord.
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