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. 2006 May 25;26(7-8):1385–1410. doi: 10.1007/s10571-006-9038-8

Nitrergic Proprioceptive Afferents Originating from Quadriceps Femoris Muscle are Related to Monosynaptic Ia-Motoneuron Stretch Reflex Circuit in the Dog

Jozef Maršala 1,3,✉, Nadežda Lukáčová 1, Dalibor Kolesár 1, Karolína Kuchárová 1, Martin Maršala 2
PMCID: PMC11520608  PMID: 16724275

Abstract

1. The aim of the present study was to examine the occurrence of the neuronal nitric oxide synthase immunoreactivity in the stretch reflex circuit pertaining to the quadriceps femoris muscle in the dog.

2. Immunohistochemical processing for neuronal nitric oxide synthase and histochemical staining for nicotinamide adenine dinucleotide phosphate diaphorase were used to demonstrate the presence of neuronal nitric oxide synthase in the proprioceptive afferents issuing in the quadriceps femoris muscle. The retrograde tracer Fluorogold injected into the quadriceps femoris muscle was used to detect the proprioceptive afferents and their entry into the L5 and L6 dorsal root ganglia.

3. A noticeable number of medium-sized intensely nitric oxide synthase immunolabelled somata (1000–2000 μm2 square area) was found in control animals in the dorsolateral part of L5 and L6 dorsal root ganglia along with large-caliber intraganglionic nitric oxide synthase immunolabelled fibers, presumed to be Ia axons. Before entering the dorsal funiculus the large-caliber nitric oxide synthase immunolabelled fibers of the L5 and L6 dorsal roots formed a massive medial bundle, which upon entering the dorsal root entry zone reached the dorsolateral part of the dorsal funiculus and were distributed here in a funnel-shaped fashion. The largest nitric oxide synthase immunolabelled fibers, 8.0–9.2 μm in diameter, remained close to the dorsal horn, while medium-sized fibers were seen dispersed across the medial portion of the dorsal funiculus. Single, considerably tapered nitric oxide synthase immunolabelled fibers, 2.2–4.6 μm in diameter, were seen to proceed in ventrolateral direction until they reached the mediobasal portion of the dorsal horn and the medial part of lamina VII. In lamina IX, only short fragments of nitric oxide synthase immunoreactive fibers and their terminal ramifications could be seen. Nitric oxide synthase immunolabelled terminals varying greatly in size were identified in control material at the base of the dorsal horn, in the vicinity of motoneurons ventrally and ventrolaterally in L5 and L6 segments and in Clarke’s column of L3 and L4 segments. Injections of the retrograde tracer Fluorogold into the quadriceps femoris muscle and cut femoral nerve, combined with nitric oxide synthase immunohistochemistry of the L5 and L6 dorsal root ganglia, confirmed the existence of a number of medium-sized nitric oxide synthase immunoreactive and Fluorogold-fluorescent somata presumed to be proprioceptive Ia neurons (1000–2000 μm2 square area) in the dorsolateral part of both dorsal root ganglia. L5 and L6 dorsal rhizotomy caused a marked depletion of nitric oxide synthase immunoreactivity in the medial bundle of the L5 and L6 dorsal roots and in the dorsal funiculus of L5 and L6 segments.

4. The analysis of control material and the degeneration of the large- and medium-caliber nitric oxide synthase immunoreactive Ia fibers in the dorsal funiculus of L5 and L6 segments confirmed the presence of nitric oxide synthase in the afferent limb of the monosynaptic Ia-motoneuron stretch reflex circuit related to the quadriceps femoris muscle.

KEY WORDS: spinal cord, dorsal root ganglia, nitric oxide synthase, monosynaptic Ia-pathway, quadriceps femoris muscle, proprioceptive neurons, dog

INTRODUCTION

Sense of position and kinesthesia are regarded as two submodalities of proprioception depending on information arising from the muscle spindles (Goodwin et al., 1972; McCloskey, 1973; Horch et al., 1975). Muscle spindles have two types of sensory endings: A primary ending supplied by a large-caliber myelinated fiber classified as group Ia and one or more secondary endings supplied by afferent fibers of intermediate size known as group II fibers (Matthews, 1964, 1977, 1981, 1982). Functionally, the primary ending is responsive to the rate of stretch of the spindle, giving a dynamic response, and to the new length, resulting in a static response. It is generally thought that the dynamic response is set, in part, by the activity of dynamic fusimotor fibers (Bessou et al., 1968). Secondary endings chiefly signal muscle length and show little dynamic responsiveness. The muscle spindle primary afferents and afferents from tendons and joints are classified as Aα fibers. These are large-caliber (15–20 μm) myelinated fibers that conduct impulses at high velocity (70–120 m/s). The muscle spindle secondary afferents are thinner (5–10 μm) and are classified as Aβ myelinated fibers that conduct impulses at lower speed (30–70 m/s; Hunt, 1954).

Based on classical descriptions, the proprioceptive and exteroceptive fibers bifurcate upon entering the spinal cord into ascending and descending branches that travel a long distance rostrally and caudally in the lateral portion of the dorsal funiculus (DF) and in the case of the proprioceptive fibers they penetrate the gray matter of the dorsal horn (DH) from its dorsomedial or medial aspect (Scheibel and Scheibel, 1969), while the exteroceptive myelinated semicircular fibers pass along the wall or within the DF and enter the gray matter underneath the substantia gelatinosa and then turn upward (Altman and Bayer, 2001). Most of the proprioceptive Ia collaterals turn ventrally and terminate in laminae VI, VII and IX, and in Clarke’s column (Brown and Fyffe, 1978). Proprioceptive Ia terminals, particularly abundant in lamina IX, form synapses with the somata and dendrites of motoneurons. Terminals around the motoneurons have the ultrastructural features of excitatory synapses (Conradi et al., 1983) and, as confirmed by punctate immnunolabelling, in all probability representing labelling of nerve terminals, glutamate-like immunoreactivity was detected in puncta apposing the motoneurons (Shupliakov et al., 1993). While physiological and EM evidence has pointed to glutamate as the fast transmitter in this synaptic connection (Maxwell et al., 1990; Örnung et al., 1995), examination of the ventral horn at high magnification has demonstrated multiple nitric oxide synthase immunoreactive (NOS-IR) varicosities and puncta in close approximation to the large somata of presumptive motoneurons; several somata were entirely surrounded by punctate NOS immunolabelling (Saito et al., 1994). Moreover, two recently identified vesicular glutamate transporters, VGLUT1 and VGLUT2, have been shown to be present in axons belonging to largely non-overlapping populations of glutamatergic neurons (Ni et al., 1995; Bellochio et al., 1998, 2000; Aihara et al., 2000; Takamori et al., 2000; Frameau et al., 2001; Sakata-Haga et al., 2001; Kaneko et al., 2002), and VGLUT1 puncta in the ventral horn while less in number were larger in size than VGLUT2 puncta and preferentially distributed to the lateral ventral horn, where they appeared to target motoneuron perikarya and dendrites (Varoqui et al., 2002).

Recently, a quantitative assessment of the distribution of glutamate-, GABA- and glycine-like immunoreactivity in boutons apposing dendrites of different caliber (range 0.2–15.0 μm) and stem dendrites in the L7 spinal cord motor nucleus revealed that 35% of the boutons were immunopositive for glutamate, 59% for GABA and/or glycine, and 6% of the boutons were judged as not enriched for any amino acid analysed (Örnung et al., 1998). Considering that most Ia boutons are an integral part of the monosynaptic Ia-motoneuron pathway and are widely variable in terms of volume, total surface area and the surface area of apposition to postsynaptic neurons, mitochondrial volume, vesicle and active zone or release site features, relation to presynaptic contacts, postsynaptic profile size, and position within the terminal arbor (Pierce and Mendell, 1993), the afferent limb of the H-reflex exemplified by structural components of the soleus-gastrocnemius muscle in the dog was reexamined immunohistochemically and it was found that the whole afferent limb of the monosynaptic Ia-motoneuron pathway, represented by proprioceptive Ia neurons with somata in L7 and S1 dorsal root ganglia (DRGs), is neuronal nitric oxide synthase immunoreactive (bNOS-IR) (Maršala et al., 2005).

In the current work, we have addressed the question of neuronal nitric oxide synthase immunopositivity in the afferent limb of the monosynaptic Ia-motoneuron pathway with special emphasis on (i) the origin of proprioceptive nitric oxide synthase immunoreactive afferents in the quadriceps femoris muscle, known to be the main antigravity acting muscle, and (ii) the course and termination of the monosynaptic Ia-motoneuron pathway in the ventrolateral part of the ventral horn (lamina IX) and lamina VII in L5–L6 segments and in Clarke’s column of L3 and L4 segments.

MATERIAL AND METHODS

Animal Model, Surgical Procedures, Tissue Sampling, Sectioning and Immunoprocessing of Sections

Adult mongrel dogs (n=12) of both sexes weighing 8–13 kg were used in these experiments. Experimental protocols were approved by the Institute of Neurobiology Animal Care Committee. The animals were divided into three groups. In the first group of control animals, the occurrence and distribution of bNOS-IR (n=3) and/or nicotinamide adenine dinucleotide phosphate diaphorase (NADPHd) exhibiting (n=3) somata and fibers in L5 and L6 DRGs, and the presence of bNOS-IR and/or NADPHd-stained fibers in the trunk of the femoral nerve (FN) and L5 and L6 dorsal roots were studied. In the same group, L5 and L6 segments were analysed with emphasis on medium- and large-caliber bNOS-IR fibers in the DF in L5 and L6 segments and their trajectory and termination in the gray matter (laminae) of both segments. In the second group (n=3), Fluorogod (FG) injections into the FN were performed, and after 4 days of survival specimens from the trunk of the FN were taken and L5 and L6 DRGs were dissected out bilaterally. Longitudinal serial sections (36 μm thick) were cut from L5 and L6 DRGs and processed alternately with FG-fluorescent microscopy and immunohistochemistry for bNOS-IR somata and fibers in L5 and L6 DRGs. In the third group (n=3), unilateral dorsal rhizotomy of L5 and L6 dorsal roots midway between the DRGs and the dorsal root entry zone (DREZ-one) was performed. For immunohistochemistry of neuronal NOS all sections cut from the femoral nerve, L5 and L6 dorsal root ganglia and L5 and L6 segments were processed for bNOS immunohistochemistry (Bredt et al., 1990) and, in general, the immunohistochemical processing was identical with that used in a recent study (Maršala et al., 2005). The sections for histochemical staining of NADPHd of the femoral nerve, L5 and L6 dorsal root ganglia and L5 and L6 segments were processed according to a modified histochemical procedure (Scherer-Singler et al., 1983; Lukáčová et al., 2006).

Tracing the Afferent Pathway from the Quadriceps Femoris Muscle to the L5 and L6 Dorsal Root Ganglia Using Retrograde Transport of Fluorogold (FG)

In the second group (n=3), retrograde tracer Fluorogold and bNOS immunohistochemistry were used to determine the occurrence and distribution pattern of proprioceptive bNOS somata in L5–L6 DRGs after microinjections of FG into the FN and quadriceps femoris muscle. In anesthetized animals, the FN was surgically approached through a midthigh incision just below the inguinal ligament, and after identification of the sartorius muscle and femoral artery the thick stem of the FN was identified. The segment of the FN extending between the branching of the saphenous nerve and small ramifications entering the vastus medialis muscle was slightly elevated, and as a rule three injections of 1.5–5.0 μL of FG solution spaced at 2 mm craniocaudally were injected into the FN. The FN below the branching of the saphenous nerve was selected owing to its pure muscular origin. The wound was then closed and animals survived for 4 days before being anesthetized with a mixture of ketamine and xylazine (100 and 15 mg/kg body weight i.m.) and intracardially perfused with heparinized saline and subsequently with freshly prepared 4% paraformaldehyde in 0.1 M phosphate-buffered saline (PBS, pH 7.4). After Th12-L4 laminectomies, the dura was opened longitudinally and after an exact verification of L1–S3 dorsal roots and corresponding DRGs the whole block, i.e. the lumbar and sacral spinal cord together with DRGs, was dissected out in toto and post-fixed at 4°C overnight and then transferred to 30% sucrose at 4°C for cryoprotection (48 h). Afterwards spinal cord segments (L1–L7 and S1–S3) and L1–L7 and S1–S3 DRGs were bilaterally excised under a dissection microscope.

L5 and L6 Dorsal Rhizotomy-Induced Depletion of Nitric Oxide Synthase Immunopositivity in the Dorsal Funiculus of L5 and L6 Segments

In the third group (n=3), L5 and L6 dorsal roots were approached and divided unilaterally as follows. The animals were anesthetized with a mixture of ketamine and xylazine (100 and 15 mg/kg b.w.) and artificially ventilated in a respirator with oxygen and nitrous oxide (Anemat N8 Chirana, ČSSR). The L5 and L6 dorsal roots were approached through lumbar laminectomy of the second to seventh laminae, thus gaining access to L5 and L6 dorsal roots and corresponding DRGs. The dorsal roots L5 and L6 were divided 2–3 mm proximally to the corresponding DRGs. Extreme care was taken to avoid damaging the radicular and spinal blood vessels. Following recovery from the anesthetic, each dog was transferred from the operating room to a holding cage for observation. Six days later the animals were deeply anethetized (as earlier) and intracardially perfused with heparinized saline and subsequently with freshly prepared 4% paraformaldehyde in 0.1 M phosphate-buffered saline (PBS, pH 7.4). The whole lumbosacral enlargement (L4–S2 segments) and corresponding DRGs were dissected out in toto, post-fixed and processed in turn in the same way as with the control material, including immunoprocessing for bNOS (Bredt et al., 1990).

The Counting of Neuronal Nitric Oxide Synthase Immunolabelled (bNOS-IR) Somata and Statistical Analysis

The number of small (<1000 μm2 square area) and medium-sized (1000–2000 μm2 square area) bNOS-IR somata was counted from 10 transverse serial sections of ipsilateral and contralateral L6 dorsal root ganglia after L5 and L6 dorsal rhizotomy. The bNOS-IR somata were counted from three animals (n=3), and their number was statistically evaluated by ANOVA as well as by the Tukey–Kramer test. Data are given as means±SEM; p<0.05 with respect to ipsilateral side.

RESULTS

The Appearance and Distribution of bNOS Immunoreactive and/or NADPH Diaphorase-Stained Somata in L5 and L6 DRGs in Control Animals

L5 and L6 DRGs contained a countable number of bNOS immunolabelled and/or NADPHd-stained cells divided according their cell body square area into small (<1000 μm2 square area), medium-sized (1000–2000 μm2 square area) and large (>2000 μm2 square area) mostly round or slightly elongated somata. There was a marked prevalence of small and medium-sized bNOS immunolabelled and/or histochemically NADPHd-stained cell bodies compared with only a slight tendency for bNOS immunolabelling or NADPHd-staining in large (>2000 μm2 square area) neurons. Although small and medium-sized bNOS immunolabelled and/or NADPHd-exhibiting neurons were found throughout the longitudinal sections of both DRGs, it was apparent that the majority of both cell categories could be detected in the dorsolateral part of L5 and L6 DRG (Figs. 1; 2A and E; 3A and E). Remarkable heterogeneity was noted in the intensity of bNOS immonolabelling and/or NADPHd-stained somata of small and medium-sized neurons. In this context, the medium-sized bNOS immunolabelled and/or NADPHd-stained neurons have been shown to exhibit a greater variability, and demonstrate some heavily bNOS immunolabelled and/or intensely NADPHd-exhibiting somata (Figs. 2B, C and F; 3B, C and F–H). Large (>2000 μm2 square area) neurons with few exceptions were found to be bNOS-immunonegative (Fig. 3C) or histochemically NADPHd-unstained (Figs. 2G; 3F and H).

Fig. 1.

Fig. 1.

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Camera lucida drawing depicting the distribution of small (<1000 μm2 square area, small dots), and medium-sized (1000–2000 μm2 square area, medium-sized dots) bNOS-IR neurons in longitudinally-cut L5 (A) and L6 (B) DRGs. Each dot represents one bNOS-IR soma; moderately labelled medium-sized bNOS-IR neurons are shown as semicircles. C, central end of the DRG; P, peripheral end of the DRG; MR, motor root.

Fig. 2.

Fig. 2.

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Microphotographs showing the distribution of bNOS immunoreactive somata and fibers (A–D) and/or NADPH diaphorase-stained somata and fibers (E–H) in the L5 DRG of the dog. (A) A low-power microphotograph of longitudinally cut L5 DRG showing the distribution of bNOS-IR somata; C, central end of L5 DRG; P, peripheral end of L5 DRG; bNOS-IR somata and fibers in boxed areas, B–D, are depicted in the following microphotographs; MR, motor root. (B) Small and medium-sized bNOS-IR somata in the dorsolateral part of the L5 DRG (boxed area B). Arrows point to intensely bNOS immunolabelled medium-sized somata; among them more lightly immunolabelled medium-sized bNOS somata (arrowheads) are seen. (C) Intensely bNOS immunolabelled medium-sized somata (arrows) and bNOS-IR fibers (asterisks) are seen in the central part of the L5 DRG (boxed area C). (D) Many intensely bNOS immunolabelled fibers (asterisks) are seen in the central part of the L5 DRG. (E) A low-power microphotograph showing a longitudinally cut L5 DRG and the distribution of NADPH diaphorase-exhibiting somata; C, central end of L5 DRG; P, peripheral end of L5 DRG; MR, motor root. Arrows point to moderately NADPHd-stained medium-sized neurons in the dorsolateral part of the L5 DRG; small intensely NADPHd-stained cell (arrowhead); NADPHd-stained fibers of varying thicknesses (asterisk) are seen. (G) Large, NADPHd-unstained somata (asterisks) are seen in the central part of the L5 DRG; the arrow points to a medium-sized moderately NADPHd-stained soma. (H) Large (arrow) and thin (arrowheads) NADPHd-stained fibers in the peripheral part of the L5 DRG close to the motor root (MR).

Fig. 3.

Fig. 3.

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Microphotographs showing the distribution of bNOS immunoreactive somata and fibers (A–D) and/or NADPHd-stained somata (E–H) in the L6 DRG of the dog. (A) A low-power microphotograph of longitudinally cut L6 DRG showing the distribution of bNOS-IR somata; C, central end of L6 DRG; P, peripheral end of L6 DRG; MR, motor root; bNOS-IR somata and fibers in boxed areas, B–D, are depicted in the following microphotographs. (B) Moderately bNOS immunolabelled (arrowheads) and one intensely bNOS immunolabelled (arrow) medium-sized neurons in the dorsolateral part of the L6 DRG; v, vessels. (C) Large (>2000 μm2 square area) bNOS immunonegative neurons (asterisks) in the central portion of the L6 DRG; arrows point to intensely bNOS immunolabelled medium-sized neurons. (D) bNOS-IR fibers of varying thicknesses (asterisks) are seen in the central portion of the L6 DRG; arrow points to a large bNOS-IR fiber; v, vessel. (E) A low-power microphotograph of longitudinally cut L6 DRG showing the distribution of NADPHd-stained somata; C, central end of L6 DRG; P, peripheral end of L6 DRG; MR, motor root. (F) Medium-sized intensely NADPHd-stained somata (arrows) and light, large non-NADPHd-stained somata (asterisks) in the dorsolateral part of the L6 DRG. (G) Medium-sized dark NADPHd-stained neurons (arrows) in the central part of the L6 DRG; arrowheads point to moderately NADPHd-stained neurons. (H) Intensely NADPHd-stained neurons (arrows); arrowheads point to medium-sized moderately NADPHd-stained neurons; large non-NADPHd-stained neurons (asterisk) are often seen in both dorsolateral and central part of the L6 DRG.

bNOS immunolabelling and/or NADPHd-staining appeared also in many intraganglionic fibers including those identified at the input (peripheral) and output (central) side of L5 and L6 DRGs (Figs. 2H and E; 3A and E). A higher accumulation of bNOS immunolabelled and/or NADPHd-stained axons was found in the central parts (Figs. 2D; 3D) than in the dorsolateral part of both DRGs. Intraganglionic bNOS-IR and/or NADPHd-exhibiting fibers of various thicknesses including large ones (8.0–9.2 μm in diameter—not counting the myelin sheath) were identified in all parts of L5 and L6 DRGs including those located close to the motor root (Fig. 2H).

A higher accumulation of intensely bNOS immunolabelled and/or NADPHd-stained fibers was consistently found at the peripheral end of L5 and L6 DRGs, and at the spinal end of L5 and L6 DRGs close to the exit of the central processes from both DRGs, an evidently lower fiber bNOS immunolabelling and/or NADPHd-staining could be detected.

The Occurrence of NADPH Diaphorase Exhibiting Fibers in the Femoral Nerve and Tracing of Afferent Pathways from the Quadriceps Muscle to L5 and L6 DRGs Using the Retrograde Transport of Fluorogold and Immunoprocessing L5 and L6 DRGs for Neuronal Nitric Oxide Synthase

Specimens of the FN were taken from the segment before the FN enters the quadriceps femoris muscle by passing between the rectus femoris and the vastus medialis muscles at the proximal end of the cleft that separates these heads. Since specimens of the FN were taken below the branching of the saphenous nerve arising from the FN before this nerve leaves the iliopsoas, no cutaneous branches were contained in the specimens of the FN subjected to histochemical analysis.

Unevenly dispersed NADPHd-exhibiting axons of varying thicknesses were found in longitudinally cut specimens of the FN. Among the thin myelinated NADPHd-stained axons, a noticeable number of large myelinated axons was intensely NADPHd-stained. While unmyelinated and thin myelinated fibers were seen loosely dispersed across the transverse section of the FN, large-caliber myelinated NADPHd-stained fibers, 8.0–9.2 μm in diameter (not counting the myelin sheath), tended to run in small microbundles containing at least 3–4 intensely stained axons (Fig. 4A and B).

Fig. 4.

Fig. 4.

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(A and B) In longitudinally cut sections through the femoral nerve (FN) processed for NADPHd histochemistry (NADPHd), single or small bundles of NADPHd-exhibiting fibers (arrows) can be seen; arrowhead points to a lightly NADPHd-stained microglia. (C and D) The arrows point to longitudinally cut FG-fluorescent axons in the trunk of the femoral nerve (FN-FG). (E) Longitudinally cut L6 DRG processed for FG-fluorescent (FG) somata after injections of Fluorogold in the femoral nerve; C, central end of L6 DRG; P, peripheral end of L6 DRG; boxed areas, G and I, are seen below at a higher magnification. (F) A consecutive section cut through the L6 DRG and processed for bNOS immunoreactivity; boxed areas, H and J, are seen below at higher magnification. (G) Enlarged upper boxed area, E/G, in the dorsolateral part of the L6 DRG containing two FG-fluorescent medium-sized somata (arrows); CA, capsule. (H) A consecutive section processed for bNOS immunoreactivity contains two identical medium-sized bNOS-IR somata; CA, capsule. (I) The enlarged lower boxed area, E/I, in the basal part of the L6 DRG (close to the motor root) contains several FG-fluorescent cell bodies; one of them is identical with the bNOS-IR soma seen in the next section; CA, capsule. (J) A consecutive section processed for bNOS immunoreactivity; an identical bNOS-IR soma (arrow) is seen in the right half above the capsule (CA). (K) Two FG-fluorescent cell bodies (boxed area in K) in the middle portion of L6 DRG; CA, capsule. (L) A consecutive section cut through the L6 DRG and processed for bNOS immunoreactivity contains two bNOS-IR cell bodies (boxed area in K) identical with the previous ones; CA, capsule.

The retrograde transport of Fluorogold injected unilaterally into the FN and quadriceps femoris muscle (vastus medialis, vastus lateralis and rectus femoris) was used to trace afferents from these muscles to L5 and L6 DRGs ipsilateral with the injections. FG-labelled afferents originating in the vastus medialis, vastus lateralis and rectus femoris muscles could be detected in the trunk of the FN taken from the portion before the fibers of the FN enter the quadriceps femoris muscle (Fig. 4C and D). Many FG-fluorescent axons were seen to enter L5 and L6 DRGs at their peripheral side. The analysis of mostly round or slightly oval-shaped FG-fluorescent cell bodies showed that the majority of FG-fluorescent somata was located in the dorsolateral (Fig. 4E, G and K) and ventral portion (Fig. 4I) of both DRGs. The square area of heavily and moderately FG-labelled medium-sized somata ranged from 1000 to 2000 μm2. The distribution pattern of some medium-sized FG-labelled neurons confirmed that a noticeable number of these neurons is immunoreactive for neuronal nitric oxide synthase (Fig. 4F, H, J and L).

The Distribution of Nitric Oxide Synthase Immunoreactive Axons in L5 and L6 Dorsal Roots and the Organization of Nitric Oxide Synthase Immnoreactive Axons in the Dorsal Funiculus of L5 and L6 Segments on Control Sections

The segregation of large-caliber bNOS-IR axons into a medial bundle and small-caliber bNOS-IR axons into a lateral bundle just before the DREZ-one was obvious considering the rootlets of L5 and L6 dorsal roots (Fig. 5A and E). The general appearance of the medial versus lateral bundle based on bNOS immunohistochemical processing is quite different. While in the normal medial bundle large-caliber bNOS-IR axons are seen as mostly heavily bNOS immunolabelled large axonal profiles 8.0–9.2 μm in diameter (not counting the myelin sheath—Fig. 5F—MB), the lateral bNOS-IR bundle entering the spinal cord and extending from the DREZ-one to the dorsal border of the dorsal horn has a characteristic punctate appearance caused by an enormous amount of bNOS-IR puncti representing in fact transversally and obliquely cut thin bNOS-IR axonal profiles (Fig. 5A, E and F). The lateral bundle then merges with punctate bNOS-IR immunopositivity detected in the superficial dorsal horn (Fig. 5B and C).

Fig. 5.

Fig. 5.

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Microphotographs showing the distribution of bNOS-IR axons in the dorsal funiculus of L5 (A–D) and L6 (E–H) segments. (A) Large bNOS-IR axons seen in the medial bundle of the L5 dorsal root (MB) pass through the dorsal root entry zone and enter the dorsal funiculus (DF); DH, dorsal horn; DLF, dorsolateral funiculus; LB, lateral bundle of the dorsal root. (B) The occurrence of large bNOS-IR axons (arrows) in the dorsolateral part of the dorsal funiculus; DH, dorsal horn. (C) Large bNOS-IR axons (arrows) in the dorsal funiculus close to the dorsal horn (DH). (D) Many bNOS-IR axons (arrows) are seen in the medial part of the dorsal funiculus along the medial border of the dorsal horn. (E) Large bNOS-IR axons of the medial bundle (MB) of the L6 dorsal root enter the dorsal funiculus through the dorsal root entry zone and are located medially to the lateral bundle (LB) of the dorsal root; DH, dorsal horn; DLF, dorsolateral funiculus. (F) Large bNOS-IR fibers (arrows) of the medial bundle (MB) are seen in the dorsolateral part of the dorsal funiculus (DF). (G) Many transversally cut bNOS-IR axons (arrows) are seen in the dorsal funiculus close to the dorsal horn. (H) Some large bNOS-IR axons (arrows) are seen in the superficial parts of the dorsal funiculus.

Large bNOS-IR axons located in the dorsomedial portion of L5 and L6 dorsal roots pass through the DREZ-one and enter the dorsolateral part of the DF (Fig. 5B and F). No preferential distribution of bNOS-IR fibers varying over a wide range from 2.5 to 9.2 μm (not counting the myelin sheath) can be discerned within the L5 and L6 DF, and bNOS-IR fibers are detectable across the upper and middle third of the DF in both segments (Fig. 5C, G and H). It is noteworthy that there are three exceptions to this description. First, the transition zone between the middle and lower third of the DF is clearly marked by a decreased packing density of the bNOS-IR axons (Fig. 5D), and second, the largest bNOS-IR axons (8.0–9.2 μm in diameter) are preferentially seen in that part of the DF which is located close to the dorsal and medial margin of the dorsal horn. Third, a narrow strip of the white matter comprising the medialmost portion of the DF is packed with extremely thin, mostly unmyelinated bNOS-IR fibers (0.8–1.5 μm in diameter).

Unilateral L5 and L6 Dorsal Rhizotomy and the Depletion of Axonal bNOS Immunopositivity in the Dorsal Root Entry Zone and Dorsal Funiculus of L5 and L6 Segments

Unilateral L5 and L6 dorsal rhizotomy was performed 2.0–2.5 mm before the beginning of the dorsal root entry into the cord. Six days post-rhizotomy, transverse sections cut from all segments of the lumbosacral intumescence (L4–S2) were processed for bNOS immunohistochemistry. Comparing the normal, non-rhizotomized with rhizotomized dorsal roots at L5 and L6 level revealed two distinct differences consisting in a marked depletion of axonal bNOS immunopositivity identified clearly in the large bNOS-IR axons located in the medial bundle (Fig. 6A and F) and adjacent portions of the DF, accompanied by anterograde degeneration traceable in a large proportion of the DF ipsilateral with the L5 and L6 rhizotomy. The phenomenon of axonal bNOS-IR depletion seen in the rhizotomized medial bundle and DF is influenced by an abrupt decrease in optical density of large bNOS-IR axons which in normal, non-axotomized neurons appear as dark brown axonal profiles (Fig. 6B, C, G and H). Moreover, a marked reduction of axonal bNOS immunolabelling following dorsal rhizotomy appeared to be due to an interruption of anterograde axoplasmic transport of bNOS putatively synthesized in the ipsilateral L5 and L6 DRGs. Given the marked reduction or almost complete loss of axonal bNOS immunolabelling, the characteristic dotted appearance of bNOS-IR axons in the dorsal, dorsomedial and medial portion of the DF ipsilateral with the L5 and L6 dorsal rhizotomy is lost and, as documented later, only irregular outlines of degenerating bNOS-depleted axons can be identified (Fig. 6G and H). The characteristic sign of anterograde axotomy-induced degeneration is accompanied by an irregular loss or decrease in packing density of myelinated bNOS-IR axons and a loss of smooth axonal outlines. Instead, many axonal distorsions or short expansions of bNOS-IR axons can be seen (Fig. 6D, E, I and J).

Fig. 6.

Fig. 6.

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Microphotographs showing the occurrence and funicular distribution of bNOS-IR axons of various thicknesses in the dorsal funiculus of the L6 segment (A–E) in control material and the depletion of axonal nitric oxide synthase immunopositivity and degeneration of bNOS-IR axons in the medial bundle of the dorsal root and in the dorsal funiculus of the L6 segment after L5 and L6 dorsal rhizotomy (F–J). (A) A low-power microphotograph showing a massive medial bundle (MB) of the dorsal root passing through the dorsal root entry zone (DREZ) and entering the dorsal funiculus (DF); D, dorsal horn; DLF, dorsolateral funiculus; LB, lateral bundle of the dorsal root. (B) Many bNOS-IR fibers (arrows) are seen in the dorsal funiculus close to the dorsal horn. (C) The distribution of bNOS-IR fibers in the dorsal funiculus close to the medial border of the dorsal horn. (D and E) Large bNOS-IR axons (asterisks) are traceable across the lateral, D, and more medial, E, part of the dorsal funiculus. (F) A low-power microphotograph showing the depletion of axonal bNOS-IR immunopositivity in the medial bundle and dorsal funiculus in the L6 segment after L5 and L6 dorsal rhizotomy; DH, dorsal horn; DLF, dorsolateral funiculus; LB, lateral bundle of the dorsal root. (G) Large, degenerating bNOS-IR axons (arrowheads) in the dorsal funiculus close to the dorsal horn. (H) The depletion of axonal bNOS immunopositivity is more clearly visible in large bNOS-IR axons (arrowheads) in the dorsal funiculus close to the medial border of the dorsal horn. (I) A high-power microphotograph of large degenerating bNOS-IR axons (arrowheads) in the dorsal funiculus after L5 and L6 dorsal rhizotomy.

Trajectory of Large bNOS-IR Fibers and Termination in the Gray Matter on Control Sections and After Unilateral L5 and L6 Dorsal Rhizotomy

Large bNOS-IR fibers entered the gray matter through the medial half of the dorsal horn and then continued into the deeper parts of the gray matter. bNOS immunohistochemistry did not confirm the existence of bNOS immunoreactive microbundles since almost all large-caliber bNOS-IR axons descended individually, and their terminal ramification areas were localized in the medial half of the base of the dorsal horn (lamina VI), and the medial part of lamina VII (Scheibel and Scheibel, 1969). The highest density of terminal ramifications and bNOS-IR boutons was found in the neuropil of the homonymous motor cell column in lamina IX of L5 and L6 segments, corresponding to the ventrolateral part of the ventral horn and reaching to its ventral border (Vanderhorst and Holstege, 1997). Most bNOS-IR boutons attached to the terminal fibers had a slightly elongated or ovoid shape, varying from 0.7 to 15.0 μm along the long axis and from 0.7 to 4.8 μm along the short axis (Fig. 7A and B). Similarly, bNOS-IR boutons greatly varying in size including giant ones were found in the neuropil of Clarke’s column in L3 and L4 segments on control material (Fig. 7C and D). The long axes of these giant bNOS-IR boutons in Clarke’s column were almost always oriented in the ventromedial direction. Some bNOS-IR boutons, mainly those found in the neuropil of Clarke’s column, were characterized by strong bNOS immunolabelling (Fig. 7C).

Fig. 7.

Fig. 7.

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(A) A high-power microphotograph showing a bNOS-IR bouton (arrow) close to a large bNOS-IR unlabelled motor neuron (asterisk) in lamina IX (LIX) of the L6 segment (L6); control material. (B) The densitogram taken from the same bouton is seen as a narrow peak sharply rising and falling with the base located in the left half of the x-axis. (C) A high-power microphotograph depicting an intensely bNOS-immunolabelled giant bouton (arrow-gbt) in Clarke’s column (C1c) in the L3 segment (L3); control material. (D) An extremely narrow peak of the densitogram in the left third of the x-axis is characteristic for an intensely bNOS-immunolabelled giant bouton in Clarke’s column. (E) bNOS-IR bouton in the intermediate zone (LVII) of the L5 segment (L5) ipsilateral with L5 and L6 unilateral rhizotomy. Centrally located depletion of bNOS immunopositivity is detectable (arrow-bt); the arrowhead points to the terminal fiber. (F) Densitogram of a partly bNOS-depleted bouton displaying two peaks and a broader base located in the left and middle third of the x-axis. (G) A high-power microphotograph of a bNOS-IR bouton (arrow-bt) with terminal fiber (arrowhead) in lamina IX (LIX) of the ventral horn in the L6 segment (L6) ipsilateral with unilateral L5 and L6 rhizotomy. Centrally located depletion of bNOS immunopositivity is visible. (H) The densitogram depicting the aforesaid bouton is characterized by a broader base located in the middle third of the x-axis. (I) A low-power microphotograph of a longitudinally cut section through the right L6 DRG (L6 DRG-R) ipsilateral with rhizotomy (rh+) and processed for bNOS immunopositivity. (J) A high-power microphotograph depicting many intensely bNOS-immunolabelled medium-sized somata (arrows) in the dorsolateral part (DL) of the L6 DRG ipsilateral with L6 rhizotomy; c-capsule. (K) Large bNOS-IR fibers (arrows) just below the dorsolateral part of the L6 DRG ipsilateral with L6 rhizotomy. (L) Intensely bNOS immunolabelled medium-sized somata (arrows) in the ventral part of the L6 DRG (vp) ipsilateral with L6 rhizotomy. (M) A low-power microphotograph of a longitudinally cut section through the left L6 DRG (L6 DRG-L) contralateral with rhizotomy (rh−). (N) Loosely dispersed small- and medium-sized bNOS-IR somata (arrowheads) in the dorsolateral part (DL) of the L6 DRG contralateral with rhizotomy (rh−).

While the extent and location of bNOS-IR depleted and degenerating axons in the DF of L5 and L6 segments after unilateral L5 and L6 dorsal rhizotomy was easily identified, large, usually single running bNOS-IR degenerating axons in the gray matter were seldom traceable for a long distance, and on transverse sections they were most often seen as short bNOS-IR axonal fragments. Unilateral L5 and L6 dorsal rhizotomy-induced depletion of bNOS immunolabelling noticed in large-caliber bNOS-IR axons of corresponding dorsal roots, in the L5 and L6 medial dorsal bundle as well as in the DF, was accompanied by changes in bNOS-IR boutons (bNOS-IRBs) located in the ventrolateral part of the ventral horn (lamina IX) and lamina VII of L5 and L6 segments and in Clarke’s column of L3 and L4 segments ipsilateral with the dorsal rhizotomy and consisting in a depletion of immunolabelling detectable in large bNOS-IRBs ranging from 6.2 to 14.5 μm in length, confirmed by densitometry (Fig. 7E, F, G and H).

L5 and L6 Dorsal Rhizotomy-Induced Upregulation in bNOS-IR in the Somata and Fibers of L5 and L6 DRGs

After unilateral L5 and L6 dorsal rhizotomy, there was a marked increase in the number of small and medium-sized bNOS imunolabelled cells in both ipsilateral L5 and L6 DRGs (Fig. 7I, J, M and N). The majority of intensely bNOS immunolabelled cells was found in the dorsolateral part of L5 and L6 DRGs, but their occurrence in the middle and ventral portion of both DRGs was not an uncommon finding (Figs. 7K and L; 8A and B; Table I). Although the quantitative assessment of the dorsal rhizotomy-induced increase in the number of bNOS-IR somata in L5 and L6 DRGs clearly demonstrates that bNOS immunolabelling is noticeably higher in the category of medium-sized bNOS-IR (1000–2000 μm2 square area) cells than that noticed in the group of <1000 μm2 square area somata, an unequivocal sign of bNOS upregulation could not be detected in large >2000 μm2 square area somata. In addition, the intraganglionic bNOS-IR fibers identified in L5 and L6 DRGs ranging from 0.8 to 8.0–9.2 μm (not counting the myelin sheath) responded to L5 and L6 dorsal rhizotomy by strongly enhancing bNOS immunolabelling well discernible in the large-caliber bNOS-IR fibers occurring at the peripheral and spinal side of both DRGs. As a rule, bNOS immunolabelled fibers were densest just below the dorsolateral part and in the central portion of both DRGs, but relatively sparse in the ventral portion close to the motor root. bNOS-IR fibers emerging from cell bodies located in the dorsolateral portion of L5 and L6 DRGs proceeded ventrally and then turned sharply in the direction of the peripheral or spinal end of both ganglia.

Fig. 8.

Fig. 8.

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Camera lucida drawing depicting the distribution of small (<1000 μm2 square area, small dots) and medium-sized (1000–2000 μm2 square area, medium-sized dots) bNOS-IR neurons in the longitudinally cut right L6 DRG (L6DRF-R) ipsilateral (A) with L5 and L6 rhizotomy and left L6 DRG (L6 DRG-L) contralateral (B) with L5 and L6 rhizotomy. Each dot represents one bNOS-IR soma; medium-sized moderately bNOS-immunolabelled neurons are shown as semicircles; C, central end of the L6 DRG; P, peripheral end of the DRG.

Table I.

The Number of Small (<1000 μm2 square area) and Medium-Sized (1000–2000 μm2 square area) Neuronal Nitric Oxide Synthase Immunolabelled (bNOS-IR) Somata in L6 Dorsal Root Ganglia in Ipsilateral (Left Row) and Contralateral (Right Row) Side after L5 and L6 Dorsal Rhizotomy

L6 DRG Ipsilateral side Contralateral side
Small-sized (<1000 μm2) 81.67±2.03 62.67±2.33*
Medium-sized (1000–2000 μm2) 37.67±5.50 19.00±1.15*
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Note. The number of bNOS-IR somata was counted from 10 serial sections of three animals (n=3) subjected to L5 and L6 dorsal rhizotomy. Transverse sections were prepared from L6 dorsal root ganglia of both ipsi- and contralateral sides. Data are given as means±SEM.*p<0.05 with respect to ipsilateral side.

DISCUSSION

The present study provides an immunohistochemical and histochemical description of the large-caliber bNOS immunoreactive axons in the femoral nerve, L5 and L6 DRGs and corresponding dorsal roots including their entry into the DF through the DREZ-one. The description of the trajectory and termination of large-caliber bNOS-IR axons presumed to be Ia fibers in the gray matter of L5 and L6 segments is given. In view of the fact that ascending collaterals of large-caliber proprioceptive fibers ensuing in the hindlimb terminate in Clarke’s column beginning at L4 level, the appearance of terminal ramification in the neuropil of Clarke’s column in L3 and L4 segments is described aswell.

Neuronal Nitric Oxide Synthase Immunopositivity in the Spinal Cord Gray and White Matter and bNOS in Anterograde Transport

Over the past decade, we among others have been interested in the distribution of bNOS immunolabelled and/or NADPHd-stained neurons in the spinal cord and DRGs, including radioassay analysis performed along the rostrocaudal axis of the spinal cord, and in the causal explanation of the origin of bNOS immunopositivity pertaining to the ventral, lateral and dorsal funiculi (Valtschanoff et al., 1992; Dun et al., 1993; Saito et al., 1994; Maršala et al., 1998, 1999, 2002, 2003, 2004, 2005; Lukáčová et al., 1999, 2003, 2005; Orendáčová et al., 2000, 2001).

Based on previous studies one can hypothesize that bNOS and nitric oxide (NO) in synaptic boutons may play a role in setting the excitability of the dorsal horn cells receiving input from skeletal muscle afferents, or, more specifically, may be involved directly in the synaptic activity. In this connection, it is not known whether or not NO exerts its effect postsynaptically, or if it is possible that it also alters the release of neurotransmitter from the presynaptic neurons (Wilson 2000). Recent findings from our laboratory support the view that NO may act through presynaptic neurons, at least in the long-projecting bulbospinal respiratory and, in fact, premotor neurons making monosynaptic contacts with phrenic motoneurons in C3–C5 segments (Maršala et al., 2002). Similarly, the putative role of NOS and NO in the presynaptic neuron was described in a long ascending premotor pathway connecting monosynaptically lumbosacral bNOS-IR neurons with motoneurons in the ventral motor nucleus in C7–Th1 segments, thus directly interconnecting the neurons of the lumbosacral intumescence with those innervating pectoralis maior and latissimus dorsi muscles (Maršala et al., 2004).

Comparing the enzyme’s activity in the upper and lower lumbar segments of the spinal cord demonstrated that cNOS activity prevailed in L4–L7 segments in the dorsal horn, and without exception catalytic nitric oxide synthase (cNOS) activity was noted in all white matter columns (Lukáčová et al., 2002) and even though a lower level of cNOS activity was found in the white matter in comparison with the gray one all along the rostrocaudal axis, the occurence of cNOS activity matter appeared to be an interesting finding suggesting that some long-projection axons or long propriospinal tracts, passing for various distances in the ventral and lateral funiculus alone or colocalized with another neurotransmitter, may reach more or less distant neuronal pools (Maršala et al., 2002, 2004). The occurrence of an exceptionally high number of bNOS-immunoreactive fibers of various thicknesses in the DF of the dog lumbosacral spinal cord leads us to reconsider the origin of bNOS-IR fibers from the corresponding DRGs, and to relate them—mainly those ranging from 8.0 to 9.2 μm in diameter (not counting the myelin sheath)—with the Ia proprioceptive afferents forming in fact the afferent limb of the stretch reflex. As explained below, detailed immunohistochemical analysis fully confirmed this hypothesis. In the present experimental paradigm, the FN and quadriceps femoris muscle of the dog were used to describe the origin, course and termination of presumed proprioceptive Ia fibers originating in this largest extensor antigravity acting muscle innervated including the proprioceptive component by the FN. It is well established that the muscle spindle primary afferents, and afferents from tendons and joints, are described as large-caliber (15–20 μm) myelinated Aα fibers conducting impulses at high velocity (70–120 m/s), while the muscle spindle secondary afferents are thinner (5–10 μm) myelinated Aβ fibers conducting impulses at lower speed (30–70 m/s). The bifurcating ascending and descending branches of Aα and Aβ fibers travel rostrally and caudally for various distances in the DF and give off collaterals that penetrate the gray matter of the dorsal horn from its dorsomedial and medial aspect (Scheibel and Scheibel, 1969).

The Occurrence of Neuronal NOS Immunopositivity in the Stretch Reflex Circuit and Corresponding Dorsal Root Ganglia

Although there is a considerable amount of data on the anatomical and electrophysiological characteristics of neuronal circuits pertaining to the stretch reflex, including presynaptic inhibition in man and experimental animals (Iles, 1986; Delwaide and Olivier, 1988; Crone and Nielsen, 1989; Crone et al., 1994; Faist et al., 1994; Nielsen et al., 1995; Mazzocchio and Rossi, 1997; Katz and Pierrot-Deseilligny, 1998; Morita et al., 2001; Dietz, 2002), there is no mention concerning the colocalization of glutamate and NOS in the primary proprioceptive Ia afferents (Maxwell et al., 1990), and despite the fact that the physiological evidence points to glutamate as the fast transmitter in the monosynaptic Ia-motoneuron pathway it remained unclear whether bNOS might occur in the afferent limb of the stretch reflex circuit (Örnung et al., 1995). The massive occurrence of bNOS-IR fibers in the DF of L5 and L6 segments found in this study necessarily poses at least two questions. First, where do these fibers come from, and second, how these fibers enter the spinal cord and terminate in the spinal gray. Quantitative analysis of myelinated bNOS-IR fibers in L5 and L6 DF revealed a broad spectrum of such fibers ranging from 1.5 to 8.0–9.2 μm (not counting the myelin sheath; Lukáčová et al., 2006). Since it is well documented that the thickness of an axon is directly related to the cell body size (McLachlan and Jänig, 1983), it should be admitted that the above-mentioned fibers originate from medium- or large-sized bNOS-IR somata in L5 and L6 DRGs.

However, an extremely small number of NADPHd-positive neurons was revealed on analyzing the segmental distribution of such neurons in all lumbar and sacral segments of the rat (Aimi et al., 1991), a finding contrasting with the relatively high number of medium-sized ones (1000–2000 μm2 cell body square area) detected not only in L5 and L6 DRGs but identified in all lumbosacral DRGs of the dog (Lukáčová et al., 2005), and those detected in the DRGs of human and rat (Terenghi et al., 1993). In the latter study, intense NOS immunolabelling was seen in several primary sensory neurons, and NOS immunoreactive cells were of the small- to medium-size type and were evenly distributed throughout the ganglia of all levels available, constituting approximately 40–50% of the whole cell population of the DRGs (Terenghi et al., 1993). Considering the number of bNOS-IR somata in DRGs in human and rat (Terenghi et al., 1993) and those identified recently in the dog, it is quite obvious that the modest number of NOS-immunolabelled DRG cells in the rat (Aimi et al., 1991) can hardly add to fiber bNOS immunoreactivity in the DF and, similarly, it appears as an inadequate source of afferents forming the dense plexus of fibers in the substantia gelatinosa containing nitric oxide synthase (Valtschanoff et al., 1992).

The discrepancy between the number of small and medium-sized NADPHd-stained somata in the rat DRGs (Aimi et al., 1991) and that found in the dog strongly suggests that inter-species differences may exist not only regarding the number of NOS-immunolabelled somata, but also in the distribution of small- and large-caliber dorsal root afferents and their termination in the spinal gray matter (Katz and Pierrot-Deseilligny, 1998). The question of the arrangement of different primary bNOS-IR afferent fibers as they approach and enter the cord through the DREZ-one should be dealt with in connection with the microtopography of the primary dorsal root afferents. Tracing of afferent pathways from the quadriceps femoris muscle and FN to L5 and L6 DRGs using the retrograde transport of FG and immunoprocessing L5 and L6 DRGs for bNOS revealed a noticeable number of FG-labelled and bNOS immunolabelled somata after unilateral injections of the retrograde tracer into the FN. FG-labelled afferent fibers ranging from 1.5 to 9.2 μm in diameter (not counting the myelin sheath) arising in the quadriceps femoris muscle were detected in the trunk of the FN and, more proximally, were seen to enter L5 and L6 DRGs at their peripheral side. It is noteworthy that the square area of most FG-labelled medium-sized somata in L5 and L6 DRGs ranged from 1000 to 2000 μm2 square area. Moreover, a detailed comparison of consecutive sections processed for FG-fluorescence and bNOS-immunolabelling confirmed that many medium-sized FG-labelled cell bodies were intensely bNOS-IR and were of the same size and location, thus pointing to a possibility that they represent a subpopulation of Ia proprioceptive DRG neurons (>soma 1000 μm2) innervating the muscle spindles and forming the afferent limb of the stretch reflex (Taylor et al., 2005).

The Presence of Neuronal Nitric Oxide Synthase Immunopositivity in the Peripheral Nerves, Medial Bundle of the Dorsal Roots and Dorsal Funiculi

Although bNOS-IR and/or NADPHd-stained fibers including coarse ones in the trunk of the dog FN appeared more or less unevenly distributed, in normal rats the fibers of the sciatic nerve were bNOS immunonegative (Gonzáles-Hernández and Rustioni, 1999a) and NADPH diaphorase staining, a histochemical marker for NOS, was virtually absent (Gonzáles-Hernández and Rustioni, 1999b). There was no detectable preferential accumulation of bNOS-IR fibers either at the peripheral or the spinal side of L5 and L6 DRGs. However, just before the beginning of L5 and L6 rootlets’ entry into the cord the segregation of large- and small-caliber bNOS-IR fibers was visible, the former being part of the medial bundle of the dorsal root, while the latter, oriented more ventrolaterally, was transformed into the lateral bundle. Both the bundles displayed distinct differences pertaining to their fiber pattern, bNOS immunolabelling, course and termination.

In an attempt to reexamine the segregation of small-caliber dorsal root axons into a lateral bundle as the rootlet enters the spinal cord (Lissauer, 1885) lumbar and high sacral dorsal roots were divided into medial coarse- and lateral fine-fiber divisions currently known as the medial and lateral bundles of the dorsal root, respectively (Ranson and Billingsley, 1916). Although the replication of these experiments confirmed that section of the lateral division abolished the potentials evoked in the midbrain tegmentum following the stimulation of the root distal to the lesion, and section of the medial division abolished the ventral thalamic potentials but left the blood pressure and tegmental-evoked potential intact (Spivy and Metcalf, 1959), detailed light and electron microscopic study of the organization of the DREZ-one in cats and monkeys indicated that a lateral division of the dorsal root is present in the monkey, but not in the cat (Snyder, 1977). It should be noted, however, that segregation into large- and small-caliber fibers, not to mention bNOS-IR axons, is also not prominent in the rat (Willis and Cogeshall, 1991). Unlike a previous study based on Ranvier’s pyridin silver technique as modified for aldehyde-fixed tissue by Earle (1952), with serial semi-fine and thin sections used for electronmicroscopic examination, the present immunohistochemical and histochemical study provides unambiguous evidence that the L5 and L6 dorsal roots just before penetrating the dorsolateral sulcus and entering the DREZ-one are not only separated into clearly discernible medial and lateral dorsal root bundles, but both the bundles also display intense bNOS immunolabelling.

The most striking finding is the occurrence of a massive bNOS-IR medial bundle extending all along the dorsolateral sulcus and the DREZ-one and contributing a substantial number of large- and medium-caliber bNOS-IR fibers to the DF of L5 and L6 segments. Passing through the DREZ-one large- and medium-caliber bNOS-IR fibers penetrate the dorsolateral part of the DF and then proceed dorsally and dorsomedially, the thickest ones being localized close to the dorsal and dorsomedial margin of the DH. Their position in the DF truly mimics the location and trajectory of impregnated Ia fibers in Golgi material identified in the DF of the cat (Scheibel and Scheibel, 1969) and the three-dimensional reconstructions of the trajectory of 23 physiologically identified Ia afferents from several hindlimb muscles of the cat (Ishizuka et al., 1979). In agreement with previous reports, the description of the differential localization of fibers in the DF (Ikeda, 1961; Uddenberg, 1968) demonstrated that large myelinated axons originating from different receptors occur in different depths in the DF, and the great majority of the large-diameter bNOS-IR axons, presumably the Ia axons described in our study, had a tendency to occur in the deep lateral and dorsal region of the DF close to the DH.

ACKNOWLEDGMENTS

The authors thank Mr. D. Krokavec, Ms. M. Špontáková, Mrs. M. Syneková, Mrs. A.M. Košová and Mrs. J. Vastag for their excellent technical assistance. The experimental work was supported by the VEGA Grant Nos. 2/3217/23 and 2/5134/25, from the SAS, APVT Grant No. 51-013002 and by NIH grants NS 32794 and NS 40386 to M.M.

Footnotes

Abbreviations ABC, avidin–biotin complex; bNOS, neuronal nitric oxide synthase; bNOS-IR, neuronal nitric oxide synthase immunoreactive; bNOS-IRBs, neuronal nitric oxide synthase immunoreactive boutons; cNOS, catalytic nitric oxide synthase; DAB, diaminobenzidine; DF, dorsal funiculus; DH, dorsal horn; DREZ-one, dorsal root entry zone; DRGs, dorsal root ganglia; eNOS, endothelial nitric oxide synthase; FG, Fluorogold; FN, femoral nerve; mNOS, macrophage nitric oxide synthase; NADPHd, nicotinamide adenine dinucleotide phosphate diaphorase; NBT, nitroblue tetrazolium; NO, nitric oxide; NOS, nitric oxide synthase; NOS-IR, nitric oxide synthase immunoreactive; PBS, phosphate-buffered saline; VGLUT1 and VGLUT2, vesicular glutamate transporters

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