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We characterized the interneurons involved in the control of ankle extensor (triceps surae [TS] muscles) motoneurons (MNs) in the lumbar enlargement of mouse neonates by retrograde transneuronal tracing using rabies virus (RV). Examination of the kinetics of retrograde transneuronal transfer at sequential intervals post inoculation enabled us to determine the time window during which only the first-order interneurons, i.e., interneurons likely monosynaptically connected to MNs (last-order interneurons [loINs]) were RV-infected. The infection of the network resulted exclusively from a retrograde transport of RV along the motor pathway. About 80% of the loINs were observed ipsilaterally to the injection. They were distributed all along the lumbar enlargement, but the majority was observed in L4 and L5 segments where TS MNs were localized. Most loINs were distributed in laminae V–VII, whereas the most superficial laminae were devoid of RV infection. Contralaterally, commissural loINs were found essentially in lamina VIII of all lumbar segments. Groups of loINs were characterized by their chemical phenotypes using dual immunolabeling. Glycinergic neurons connected to TS MNs represented 50% of loINs ipsilaterally and 10% contralaterally. As expected, the ipsilateral glycinergic loINs included Renshaw cells, the most ventral neurons expressing calbindin. We also demonstrated a direct connection between a group of cholinergic interneurons observed ipsilaterally in L3 and the rostral part of L4, and TS MNs. To conclude, transneuronal tracing with RV, combined with an immunohistochemical detection of neuronal determinants, allows a very specific mapping of motor networks involved in the control of single muscles. J. Comp. Neurol. 519:3470–3487, 2011. © 2011 Wiley-Liss, Inc.

Although it is known that each spinal cord segment receives thin-fibre inputs from several segmental dorsal roots, it remains unclear how these inputs converge at the cellular level. To study whether C- and Aδ-afferents from different roots can converge monosynaptically on to a single substantia gelatinosa (SG) neurone, we performed tight-seal recordings from SG neurones in the entire lumbar enlargement of the rat spinal cord with all six segmental (L1–L6) dorsal roots attached. The neurones in the spinal cord were visualized using our recently developed oblique LED illumination technique. Individual SG neurones from the spinal segment L4 or L3 were voltage clamped to record the monosynaptic EPSCs evoked by stimulating ipsilateral L1–L6 dorsal roots. We found that one-third of the SG neurones receive simultaneous monosynaptic inputs from two to four different segmental dorsal roots. For the SG neurones from segment L4, the major monosynaptic input was from the L4–L6 roots, whereas for those located in segment L3 the input pattern was shifted to the L2–L5 roots. Based on these data, we propose a new model of primary afferent organization where several C- or Aδ-fibres innervating one cutaneous region (peripheral convergence) and ascending together in a common peripheral nerve may first diverge at the level of spinal nerves and enter the spinal cord through different segmental dorsal roots, but finally re-converge monosynaptically on to a single SG neurone. This organization would allow formation of precise and robust neural maps of the body surface at the spinal cord level.

Spinal neurons and networks that generate rhythmic locomotor activity remain incompletely defined, prompting the use of molecular biological strategies to label populations of neurons in the postnatal mouse. During spinal cord development, expression of Lhx3 in the absence of Isl1 specifies a V2 interneuronal fate. In this study, postnatal V2-derived interneurons were identified by yellow fluorescent protein (YFP) expression in the double-transgenic offspring of Lhx3Cre/+ x thy1-loxP-stop-loxP-YFP mice. While some motoneurons were labelled, several populations of interneurons predominantly located in lamina VII could also be distinguished. Small interneurons were located throughout the spinal cord whereas larger interneurons were concentrated in the lumbar enlargement. Some V2-derived interneurons were propriospinal, with axons that bifurcated in the lateral funiculus. V2-derived interneurons gave rise to populations of both excitatory and inhibitory interneurons in approximately equal proportions, as demonstrated by in situ hybridization with VGLUT2 mRNA. Immunohistochemical studies revealed YFP+ boutons throughout the spinal cord. Both glutamatergic and glycinergic YFP+ boutons were observed in lamina IX where many apposed motoneuron somata. GABAergic YFP+ boutons were also observed in lamina IX, and they did not form P-boutons. At P0, more than half of the YFP+ interneurons expressed Chx10 and thus were derived from the V2a subclass. In adult mice, there was an increase in Fos expression in V2-derived interneurons following locomotion, indicating that these neurons are active during this behaviour. The heterogeneity of V2-derived interneurons in adult mice indicates that physiologically distinct subpopulations, including last-order interneurons, arise from these embryonically defined neurons.

The mechanism of end-to-side neurorrhaphy is believed to be by collateral sprouting, although evidence for this is lacking. This study validates whether axonal sprouting originates from the donor intact nerve by collateral sprouting with the use of a fluorescent double-labeling technique. End-to-side neurorrhaphy was performed on adult female Sprague-Dawley rats. Eight and 12 months postoperatively, animals were injected with true blue and diamidino yellow into the tibialis anterior and/or gastrocnemius muscles and were transcardially perfused with fixative after 7 days of retrograde transport. The lumbar enlargement and the dorsal root ganglia from L3 to L6 were harvested and serial sectioning and fluorescent microscopy were performed. No double-labeling neurons were observed in control animals, but a group of neurons that were greenish or yellowish in color were seen with single labeling. Double-labeling neurons, however, were seen in animals treated with end-to-side neurorrhaphy whether or not perineurotomy was performed. These results demonstrate that one parent nerve fiber can emanate another axon by collateral sprouting following end-to-side neurorrhaphy. We hypothesize that the causes of collateral sprouting might result from “switching signals” and/or “switching factors.” © 1999 Wiley-Liss, Inc. MICROSURGERY 19:281–286 1999

Previous physiological and behavioral studies have shown that the nucleus raphe obscurus (nRO) modulates pelvic floor reflex function (Yamanouchi and Kakeyama [1992] Physiol. Behav. 51:575–579; Beattie et al. [1996] Soc. Neurosci. Abstr. 22:722.4; Holmes et al. [1997] Brain Res. 759:197–204). In the present study, small injections of fluorescent tracers were used to investigate direct descending projections from the rostral and caudal portions of the brainstem nRO to retrogradely labeled pudendal motoneurons (MN) in the male rat. The caudal nRO projects into the ventral and lateral funiculi of the spinal cord, with arborizations in the thoracic intermediolateral cell column; in laminae VII, IX, and X of the lumbosacral cord; and in the sacral parasympathetic nucleus (SPN). Many identified external anal sphincter and ischiocavernosus MNs appeared to be in direct apposition with fibers originating from the caudal nRO; and more than half of the bulbospongiosus MNs that were identified appeared to receive such descending input. In addition to the nRO spinal autonomic and pudendal motoneuronal targets, projections were observed to regions of the intermediate gray that contain interneurons organizing the pelvic floor reflexes and to MN pools that are involved in functionally related somatic activities. Finally, several neurons in the lumbar enlargement were labeled retrogradely with FluoroRuby after injections into the nRO and the immediately adjacent reticular formation. Thus, the nRO may be in a position to modulate the coordinated actions of autonomic preganglionic and functionally related skeletal MN activity involved in sexual and eliminative reflex functions. J. Comp. Neurol. 397:458–474, 1998. © 1998 Wiley-Liss, Inc.

The organization of neurons in the lumbar enlargement of the rat spinal cord processing information conveyed by group II afferents of hind-limb muscle nerves has been investigated by using cord dorsum and intraspinal field potential recording. Group II afferents of different muscle nerves were found to evoke their strongest synaptic actions in specific segments of the lumbar cord. Group II afferents of quadriceps and deep peroneal nerves evoked potentials mainly at the rostral end of the lumbar enlargement (L1-rostral L3), whereas group II afferents of gastrocnemius-soleus and hamstring nerves evoked their main synaptic actions at the caudal end of the lumbar enlargement (L5). In the central lumbar segments (caudal L3‐L4), the largest group II potentials were produced by afferents of tibialis posterior and, to a lesser degree, flexor digitorum longus. Field potentials evoked by group II afferents of quadriceps, tibialis posterior, and flexor digitorum longus were largest in the dorsal horn (up to 600 µV), but also occurred in the ventral horn where they were sometimes preceded by group I field potentials. In contrast, field potentials evoked by group II afferents of gastrocnemius-soleus and hamstring nerves were restricted to the dorsal horn. These results indicate that neurons in different segments of the rat lumbar spinal cord process information from group II afferents of different hind-limb muscles. Furthermore, the topographical organization of group II neuronal systems in the rat is similar in several respects to that in the cat and may therefore represent a general organizational feature of the mammalian spinal cord. J. Comp. Neurol. 394:357‐373, 1998. r 1998 Wiley-Liss, Inc. Indexing terms: muscle afferents; motor control; motoneurons; interneurons; field potentials

β,β′-iminodipropionitrile (IDPN) produces ‘spheroids’ similar to those in certain cases of amyotrophic lateral sclerosis (ALS). Therefore, the target molecule of IDPN could be important to the understanding of the molecular mechanism of spheroid formation in ALS. Wistar rats were injected ip with 14C-labeled IDPN (14C-IDPN) and killed at 0.5, 1, 3, 6, 12 and 24 h thereafter. The radioactivity in each organ increased rapidly and reached the maximum at 0.5–1 h after 14C-IDPN injection. Thereafter, a rapid decrease occurred until 6 h, followed by a gradual decline until 24 h. The radioactivity in the cerebral cortex, diencephalon and cerebellum was higher than in the pons, medulla oblongata and spinal cord. Although high in the visceral organs and skeletal muscles, no or little radioactivity was detected in fat tissue. Autoradiography also confirmed these results. In three rats, 14C-IDPN was injected to the lumbar enlargement of the spinal cord. Six hours after injection, the segment was removed and homogenized with physiological saline (PS). After centrifugation, the supernatant was obtained (PS fraction). The pellet was resuspended with 4 mol/L urea and the supernatant was obtained (urea fraction). Each fraction was analysed by gel filtration. A peak of radioactivity was observed at the elution fraction Nos 19 and 20 (consistent with free 14C-IDPN) when PS fraction was applied. On application of urea fraction, another peak was obtained at the elution fractions Nos 8 and 9 (MW 60∼80 kDa). The present study demonstrates that 14C-IDPN does not selectively accumulate to the spinal cord and suggests that an IDPN-binding molecule with an MW of 60–80 kDa is present in the spinal cord. The molecule may be related to the pathological process of spheroid formation in ALS.

Stimulation of trigeminal afferents has been reported to have powerful effects on the spinal cord in adult animals of several species. In the present study, the pathway transmitting these influences was investigated in the neonatal rat. Experiments were performed on in vitro brainstem/spinal cord preparations. Stimulation of the trigeminal nerve evoked bilateral polysynaptic discharges in lumbar ventral roots. Intracellular recordings from lumbar motoneurons showed mainly excitatory responses, although a few inhibitory responses were also observed. Experiments with perfusion of different parts of the preparation with general or selective synaptic blockers revealed a synaptic relay under GABAergic control in the brainstem, and at least one synapse in the cervical and in the thoracic spinal cord. The involvement of lumbar interneurons was established by perfusing the lumbar enlargement with saline containing either a high concentration of divalent ions or mephenesin in order to reduce transmission along polysynaptic pathways. The contribution of excitatory amino acid transmission was evaluated and was found to evoke mixed receptor responses. The course of the pathway was traced by using different lesions to the brainstem and spinal cord. The pathway was found to be ipsilateral in the brainstem and to become bilateral in the spinal cord. The results of the present study demonstrate that polysynaptic sensorimotor pathways are present at birth. The results are discussed in relation to the pontomedullary locomotor strip, which has been thought to share many features with the trigeminal system.

The distribution of the retinoid-inducible enzyme, tissue transglutaminase (tTG) in developing rat spinal cord was determined by enzyme assay and immunocytochemistry. tTG activity was at its highest in the forebrain in late foetal development. In hindbrain and spinal cord, elevated activity persisted until after birth. In spinal cord only, a second peak of activity occurred during the first week post partum (P3). tTG was associated with both the cytosolic and particulate tissue fractions throughout spinal cord development, but the particulate component was more prominent in the early postnatal period. tTG was more concentrated during this period in the ventral horn, where the particulate-associated enzyme activity was highest. In spinal cord at 3 days post partum, particulate tTG could be solubilized with lubrol-PX, dithiothreitol and potassium thiocyanate. Both soluble and particulate-associated tTG coeluted with guinea-pig liver transglutaminase C by DEAE-sephacel chromatography. The first peak of tTG activity during late foetal life coincided with the transient localization of the enzyme by immunocytochemistry in vascular endothelia throughout the spinal cord. The second peak of activity at 3 days post partum, by which time vascular immunoreactivity was absent, coincided with the occurrence of small numbers of intensely immunoreactive motor neurones in the ventral horn. Immunoreactive motor neurones were seen predominantly at two levels: the lower thoracic segments and lumbar enlargement. The abnormal appearance of many immunoreactive neurones suggested degenerative changes were occurring. tTG was also present in central canal cluster cells from birth onwards. No neuronal immunoreactivity was seen throughout foetal development. A proportion of motor neurones prepared from E15 spinal cord and grown in coculture with spinal cord astrocytes, were immunoreactive for tTG. All immunoreactive neurones showed signs of degeneration. Addition of myotube-conditioned medium (a source of cholinergic differentiation factor, CDF) reduced the proportion of tTG-immunoreactive neurons in the cultures. Schwann cell-conditioned medium (a source of ciliary neurotrophic factor, CNTF) had a similar but less potent effect on the numbers of immunoreactive neurones. The possibility that tTG is a marker for late, but not early-phase programmed cell death in the developing rat spinal cord is discussed in the light of a proposed role for tTG in the mechanism of natural cell death by apoptosis.

The central arborizations in the dorsal horn of the spinal cord of 23 rapidly adapting (RA) A-beta primary afferent neurons innervating different regions of the glabrous skin of the hindpaw were studied by the intra-axonal injection of horseradish peroxidase in adult rats. A total of 284 arbors of the complex, simple, and blind-ending variety were recovered. The arbors of RA afferents innervating the toes, paw pads, and non-pad hindpaw differed from each other in branch pattern and dimensions. The simple and complex arbors, which are both bouton-containing, were distributed mainly in laminae III-V, although some complex arbors projected dorsally into lamina IIi. The hindpaw glabrous skin afferent terminals were located in the lumbar enlargement from caudal L3 to rostral L6. A crude somatotopic organization was observed such that toes 1-5 were represented successively in more caudal positions from mid-L4 to caudal L5. The paw pads were organized in a rostrocaudal sequence moving from the paw pads proximal to toe 1 across the foot to the paw pads proximal to toe 5, from caudal L3 to mid-L5. Non-pad hindpaw afferents were located in caudal L5. Overlap between toe, paw pad and non-pad afferent central fields was present, however, and the central terminals of afferents with non-adjacent peripheral receptive fields were shown to occupy the same region of the dorsal horn.

Cut dorsal root axons regenerate into intraspinal transplants of fetal spinal cord and establish synaptic connections there. The aims of the present study were to describe the progression of dorsal root growth within the transplants and the maturation of transplant morphology and to determine whether the regenerated dorsal root axons persist within the transplants or eventually withdraw. Embryonic (E) day 14 spinal cord was grafted into the lumbar enlargement of adult Sprague-Dawley rats, and the L4 or L5 dorsal root was cut and juxtaposed to the transplants. The morphology of the transplants was examined from 1 day to over 1 year after surgery, and the regenerated dorsal roots were labeled with immunohistochemical methods to study the subset that contains calcitonin gene-related peptide (CGRP). Embryonic spinal cord transplants survived and grew within the host spinal cord in over 90% of the animals. Transplant volume increased and the morphology of the transplants matured over the first 12 weeks and then did not change for 48–60 weeks. During the first week the transplants were composed of dissociated neurons, glia, and hematogenous cells with considerable extracellular space between them. Subsequently, the grafted neurons became densely aggregated, and non-neuronal elements such as inflammatory cells and myelin debris disappeared. CGRP-immunoreactive dorsal roots began to regenerate into the transplants within 24 hours, formed dense bundles by 4 days, and were still present at 60 weeks, the longest survival period examined. Myelination of axons within transplants began at 2 weeks. Quantitative analysis showed that the area of the transplants occupied by CGRP-labeled axons and the distribution area of the labeled axons within the transplants increased until 12 weeks and persisted unchanged for over 48 weeks. These results indicate that regenerated dorsal root axons are permanently maintained within transplants of embryonic spinal cord and suggest that the transplants can contribute to the permanent restoration of damaged intraspinal neural circuits. © 1992 Wiley-Liss, Inc.

During development, nerve growth factor (NGF) regulates the density and character of peripheral target innervation (Barde, Neuron, 2, 1525 - 1534, 1989; Ritter et al., Soc. Neurosci. Abstr., 17, 546.2, 1991); its role in adult animals is less well defined. Here we have asked if the availability of growth factors such as NGF in peripheral tissues can influence the pattern of primary afferent connections in the CNS. Using osmotic minipumps, we raised the levels of NGF in rat skeletal muscle in vivo, a tissue where the levels of this factor are normally very low (Korsching and Thoenen, Proc. Natl. Acad. Sci. USA, 80, 3513 - 3516, 1983; Shelton and Reichardt, Proc. Natl. Acad. Sci. USA, 81, 7951 - 7955, 1984; Goedert et al., Mol. Brain Res., 1, 85 - 92, 1986). After 2 weeks of treatment we asked if the sensory neurons innervating this tissue showed an altered strength and distribution of connections with dorsal horn neurons. The contralateral (vehicle-treated) muscle, and totally untreated animals, served as controls. In normal and vehicle-treated animals, electrical stimulation of muscle afferents excited relatively few neurons in the dorsal horn, and these generally showed only weak responses. In contrast, on the NGF-treated side many more dorsal horn neurons in the lumbar enlargement of the spinal cord were excited by muscle afferents. The increased responsiveness could not be explained by a generalized increase in dorsal horn excitability, since spontaneous activity was not enhanced, nor by a change in A-fibre-mediated inhibitions from the treated afferents. Thus, these afferents appeared to establish new synaptic connections or strengthened previously weak ones as a result of increased neurotrophic factor availability. The data suggest that, in the adult rat, the levels of growth factors in peripheral targets may be used to regulate an appropriate degree of afferent connectivity within the central nervous system.

Descending supraspinal and propriospinal neurons projecting to the female rat sacrocaudal spinal cord, the portion of the spinal cord that innervates the tail, were identified following injection of Fluoro-Gold into the S1-Ca2 spinal cord segments. This study attempted to determine anatomical substrates for propriospinal and supraspinal control of the tail. Propriospinal neurons were identified throughout laminae V-VIII and X at all levels of the spinal cord. The greatest density of labeling was in the lumbar enlargement, followed by the cervical enlargement, with least in the thoracic spinal cord. Within a given cord level, labeling was greatest within the intermediate zone. In addition, other prominent spinal cord collections included neurons in 1) lamina V of the lumbar enlargement, 2) dorsal lamina X of the cervical enlargement, and 3) the lateral spinal nucleus within the cervical enlargement. Supraspinal cells were identified within raphe nuclei, reticular formation nuclei, dorsal column nuclei, vestibular nuclei, noradrenergic groups, the red nucleus, the periaqueductal gray, the hypothalamus, and the motor cortex. These data indicate that there are significant descending projections to the sacrocaudal spinal cord, with distributions similar to those of other cord levels. Functionally, important supraspinal and propriospinal influences on tail, pelvic viscera and limbs, such as with locomotion, balance, defense, micturition, defecation, and sexual functions, may be mediated by these connections.

Many neurotransplantation studies have dealt with the ability of solid fetal spinal grafts to develop in the previously traumatized spinal cord of a host. In neurodegenerative spinal diseases, however, motoneuronal death occurs in the absence of a trauma, i.e., in the absence of axotomy of afferent fibers. Lesioning the spinal cord with an excitotoxic agent may provide a useful neurodegenerative model. The present study has been undertaken to determine whether homotypic fetal neurons transplanted as a cell suspension are able to rebuild a neural circuitry. Emphasis is given here to the analysis of the development of transplanted motoneurons and host-graft connectivity. The lesion was made by kainic acid on the right side of the lumbar enlargement 1 week before transplantation. The fetal spinal cords were taken from rat embryos (gestational day E12-13) and transplanted as cell suspensions. Light- and electron-microscopic analysis demonstrated that the excitotoxic lesion extended over the entire spinal segment and was confined primarily to the ventral and intermediate horns, implying the death of all motoneurons with consequent paralysis and muscular atrophy of corresponding hindlimb. The lesion was characterized by a lack of neurons, glial proliferation, and sparing of fibers of passage and afferents. Two to fourteen months after surgery, the transplants were generally large, occupying most of the neuron-depleted area. The boundaries between the transplant and host tissue were clearly delineated by the higher cellular density of the graft and the particular cytoarchitecture, i.e., the cell suspension grafts did not display a laminar organization. Among the different neuronal populations within the transplant, one resembled motoneurons: large, typically Nissl-stained and immunoreactive for calcitonin gene-related peptide (CGRP). No grafted neuron, however, extended an axon into the host ventral roots. Monoaminergic afferents from the host were studied using immunostaining for serotonin, noradrenaline, and tyrosine hydroxylase. These afferent fibers, thin and varicose, grew for a long distance and formed a network within transplants. Similarly, primary sensory CGRP-immunoreactive fibers (entering the graft from the dorsal host-graft interface) penetrated deeply into transplants. The response of cortico-and rubro-spinal afferents to the implantation of fetal tissue was different. After injection of WGA-HRP, a few anterogradely labeled cortical and rubral fibers entered only the most peripheral portion of transplants. In conclusion, our results indicate that fetal spinal neurons can be successfully transplanted into the adult neuron-depleted spinal cord. Host-to-graft connections can be formed, although their spatial extent in the transplants may depend upon structural features of the afferent fiber systems. In contrast, axonal growth of transplanted neurons into the depopulated ventral roots was not observed. Such a failure may be due to the nonpermissive substrate formed by mature oligodendrocytes and myelin of the spinal white matter.

Collateral projections are an important feature of the organization of ascending projections from the spinal cord to the brain. Primate spinothalamic tract (STT) neurons with collaterals to the periaqueductal gray (PAG) were studied by means of a fluorescent double-labeling method. Granular Blue and rhodamine-labeled latex microspheres were placed in the ventral posterior lateral (VPL) nucleus of the thalamus and the periaqueductal gray, respectively. Single and double labeled neurons were studied in the upper cervical cord, cervical enlargement, thoracic cord, lumbar enlargement, and sacral segments. The laminar distribution of double labeled neurons was similar to that of spinomesencephalic tract (SMT) neurons. Most double labeled (STT-SMT) neurons were located in contralateral laminae I, V, VII, and X. Relatively more lamina I STT-SMT neurons were found in the cervical enlargement and more lamina V STT-SMT neurons in the lumbar enlargement. The density of STT-SMT neurons in the upper cervical segments and cervical enlargement was almost equal. The density of STT-SMT neurons in the lumbar enlargement was 40% of that in the cervical enlargement. The thoracic and sacral segments had the lowest density of STT-SMT neurons, about 10% of that in the cervical enlargement. STT-SMT neurons constituted 14.7% of SMT neurons and 6% of STT neurons in the cervical enlargement and 15.3% of SMT neurons and 2.9% of STT neurons in the lumbar enlargement. The branch points of eight STT-SMT axons were studied electrophysiologically. The average percentage of conduction time spent in the parent axon was more than 85% for an antidromic action potential from the VPL nucleus and 91% from the PAG. Branch points of STT-SMT axons were calculated to be 9–13 mm caudal to the PAG, in the pons or rostral medulla.

The spinal cord is a glucocorticoid-responsive tissue, as demonstrated by hormonal effects on enzyme induction and by the presence of type II and type I glucocorticoid receptors in cytoplasmic extracts of this CNS region. Using microdissection techniques, we have found in the present investigation that glucocorticoid type II receptors are the most abundant class detected in gray (ventral and dorsal horns) and white (lateral funiculus) matter and that the distribution of type II sites among these regions was quantitatively similar. Type I sites were also quantified, with a slight prevalence in gray matter as opposed to white matter. Furthermore, stimulation of an inducible enzyme, ornithine decarboxylase (ODC), was found in ventral horn and lateral funiculus but not in dorsal horn after administration of dexamethasone (DEX), a type II receptor ligand. We also found that surgical transection of the spinal cord, while markedly increasing ODC activity per se, did not prevent the stimulatory effect of DEX administration on ODC activity measured in the lumbar enlargement of the spinal cord located below the surgical lesion. Taken together, the results suggest a direct effect of glucocorticoids on ODC activity in the spinal cord of rats, probably mediated by glucocorticoid receptors (type II) found in target cells of the ventral horn and lateral funiculus. The results also indicate that glucocorticoid receptors of the dorsal horn were not involved in ODC induction, and a function for these receptors awaits the results of further experimentation.

Nociceptin (N/OFQ), possibly acting through its receptor (OP4), shows anti-nociceptive properties when administered in the spinal cord of neuropathic rats (Yamamoto et al., 1998; Hao et al., 1999). OP4 has also been described as a mediator of spinal facilitation occurring in the chronic constriction injury (CCI) rat model (Yamamoto et al., 2000). Thus, we studied the spinal effect of N/OFQ in this model and the molecular changes that occurred in both lumbar enlargement and dorsal root ganglia (DRG) of neuropathic rats. The sciatic nerve of rodents was unilaterally ligated following the Bennett & Xie (1988) procedure and their mechanical pain threshold was recorded by von Frey hairs. Rats responding to non-noxious stimuli (

Large, mediolaterally oriented neurons in lamina I of the spinal cord, frequently referred to as marginal cells of Waldeyer, are known to project to supraspinal targets via the anterolateral tract (ALT). Although dendritic organization of lamina I neurons has been extensively studied, little is known about their local axonal morphology and branching. With the help of oblique illumination, we visually identified large lamina I neurons in the isolated lumbar enlargement (L1–L6) of the spinal cord of P14–P20 rats. By using intracellular and cell-attached biocytin injections, we achieved extensive axonal and dendritic labeling in 77 lamina I cells, 40 of which were identified as ALT projection neurons. In the majority of the cases (n = 28), the main axon of these projection neurons gave rise to one or more thin collaterals on the ipsilateral side. Based on their trajectory and location, these collaterals could be divided into three major categories: dorsal, lateral, and ventral. Lamina I projection neurons had dorsal (n = 5), lateral (n = 8), or ventral (n = 6) collaterals only or a combination of these collateral types (n = 9). Our results suggest that lamina I ALT projection neurons can additionally function as local-circuit and propriospinal neurons participating in intra- and intersegmental spinal cord processing. J. Comp. Neurol. 518:2645–2665, 2010. © 2010 Wiley-Liss, Inc.

With the multiple fluorescent retrograde tracer technique, the collateralization in the spinal cord of descending supraspinal pathways was studied in a lizad, Varanus exanthematicus. Fast Blue (FB) gels were implanted unilaterally in the spinal gray matter of the cervical enlargement and Nuclear Yellow (NY) gels were implanted ipsilaterally in two series of experiments in all spinal funiculi of the lumbar enlargement or in midthoracic spinal segments, respectively. All brainstem nuclei known to project to the spinal cord in reptiles were found to give rise to branching axons that may influence widely separate levels of the spinal cord. The number of double-labeled FB-NY cells varied in these brainstem nuclei from none to half the number of neurons projecting to the cervical enlargement. Highly collateralizing projections (expressed as the percentage of double-labeled neurons, DL) were found to arise from the nucleus raphes inferior, the contralateral nucleus reticularis superior pars lateralis, the contralateral nuclei vestibulares ventromedialis and descendens, and the ipsilateral nucleus reticularis inferior pars ventralis. A lower percentage of DL neurons was noted for the contralateral nucleus ruber and bilaterally for the nucleus reticularis medius and nucleus reticularis inferior. Extensive brainstem projections directed to cervical and high thoracic spinal levels originate from the area lateralis hypothalami, the nucleus of the fasciculus longitudinalis medialis, the contralateral nucleus cerebellaris medialis, and from the nucleus tractus solitarii. Projections preferentially directed to midthoracic or lower levels of the spinal cord were found to arise from the ipsilateral locus coeruleus, the contralateral nucleus reticularis superior pars lateralis, the nucleus reticularis inferior pars ventralis, the nucleus reticularis inferior, and the nucleus raphes inferior. In contrast to findings in mammals, in Varanus exanthematicus the red nucleus, the nucleus vestibularis ventrolateralis, and certain parts of the reticular formation did not display a clearcut somatotopic organization. In general two different patterns of collateralization can grossly be discerned: (1) a gradual decrease of spinal coaterals caudalward, which can be interpreted as a certain specificity of such projections; and (2) a constant number of collateral nerve fibers throughout the spinal cord that can be interpreted as either a nonspecific or, in contrast, a highly specific system, focussed exclusively on the cervical and lumbar enlargements.

The antinociceptive action of mesaconitine (MA) microinjected into the nucleus reticularis gigantocellularis (NRGC), the nucleus reticularis paragigantocellularis (NRPG), the periaqueductal gray (PAG) or the lumbar enlargement was investigated in rats by use of the tail immersion test. In addition, the effects of beta-adrenoceptor antagonists and an alpha-adrenoceptor antagonist administered intrathecally (i.t.) on the antinociceptive action of MA given into the NRPG were also examined by the tail immersion test. MA (50, 100 ng per rat) microinjected into the NRGC, the NRPG, and PAG and the lumbar enlargement increased the response latency in rats in a dose-dependent fashion. MA (50 ng per rat) microinjected into neighbouring sites, the nucleus reticularis parvocellularis, the nucleus originis nervi abducentis and the fasciculus longitudinalis medialis, elicited no significant effect. Intrathecally administered propranolol (1 and 5 micrograms per rat), atenolol (1 and 5 micrograms per rat) and IPS-339 (1 and 5 micrograms per rat) remarkably inhibited the increase of the response latency induced by MA (50 ng per rat) given into the NRPG. Intrathecally administered phenoxybenzamine (1 and 5 micrograms per rat) inhibited the increase of the response latency induced by MA (50 ng per rat) injected into the NRPG but to a lesser extent than the beta-adrenoceptor antagonists. It is concluded that the NRGC, the NRPG, the PAG and the lumbar enlargement are involved in the sites of the antinociceptive action of MA and that the antinociceptive effect of MA administered into NRPG is elicited by activation of the inhibitory noradrenergic neurones from the NRPG in particularly via beta-receptor-mediated effects of noradrenaline.

Projections from the spinal cord and the dorsal column nuclei (DCN) to the ventrobasal complex of the thalamus (VB) were studied in the rat by using double anterograde labeling strategy. This strategy was based on the injection of 3H-leucine into the DCN and of wheat germ agglutinin conjugated to horseradish perexidase (WGA-HRP) into the spinal cord and their subsequent transport. Adjacent 30-μm-thick sections were then processed differentially for autoradiography or for HRP by using tetramethyl benzidine (TMB) as a chromogen. Similar areas of the ventrobasal complex were labeled, in adjacent sections, after a large injection of 3H-leucine into the DCN and when wheat germ agglutinin-HRP had been injected in any part of the spinal cord. If, however, a small injection of the radioactive tracer was centered in the gracile nucleus and compared with an injection of WGA-HRP placed in the lumbar enlargement of the cord, the rostral and dorsal portions of the lateral VB were labeled from both sources. On the other hand, if tritiated leucine was injected into the cuneate nucleus, and WGA-HRP placed in the cervical enlargement, then the caudal and ventral portions of the lateral VB demonstrated overlap of both labels. The present results show that, in the rat, areas of termination of both the spinothalamic tract and the lemniscal pathway originating from the DCN overlap in the lateral VB. This overlap is somatotopically organized, thus indicating that the same area of the VB receives somatic inputs from one particular part of the body through both pathways. These results are discussed in comparison to those of comparable studies performed in the cat and in the monkey and with reference to the electrophysiological data that have demonstrated that, in the rat VB, neurons responding to noxious stimulation are intermingled with neurons exclusively responding to non-noxious stimulation.

Studies using axonal transport techniques on the North American opossum show that rubral neurons innervating the cervical cord are not distinctly separated from those which project to lumbar levels. This absence of clear rubrospinal somatotopy contrasts with that described for the placental mammals studied to date. Use of fluorescent markers in double-labelling experiments shows that most rubral neurons in the opossum still innervate either the cervical or lumbar enlargement alone, but that some supply collaterals to both levels.

The method of retrograde axonal transport of horseradish peroxidase (HRP) was used to identify the locations of cells of origin of the spinothalamic tract in the cat. Injections of from 0.2–3.0 μl of 30% HRP were made unilaterally in various regions of the somatosensory thalamus. Massive injections of the caudal thalamus in several cats showed the spinothalamic cells of origin to be located mainly in laminae I, VII and VIII in the lumbar enlargement, and in laminae I, V and VII–VIII in the cervical enlargement. Small injections of HRP were made into the three major spinothalamic terminal zones in the thalamus, to determine the laminar origin(s) of the spinal projections to each zone. Neurons in lamina I in both cervical and lumbar enlargements were found to project almost exclusively to the rostral VB-caudal VL border zone. A small number of neurons in laminae VII and VIII also project there but a larger number project to the intralaminar region. Neurons projecting to the PO regions were located mainly in laminae IV and V. This anatomical segregation of thalamic afferents probably reflects a functional segregation of input, since the functional properties of spinal neurons vary according to their laminar location. Comparison of these data with the differential projection spinothalamic neurons in the rat and monkey indicate that it is unlikely that the proposed “paleo-” and “neospinothalamic” systems would arise from anatomically separate groups of spinal neurons.

1. In unanaesthetized rats, intrathecal injection of 0.30 mumole noradrenaline (NA) at the level of the lumbar enlargement produced a transient rise in core temperature followed by prolonged hypothermia and tail skin vasodilation.2. Studies of the distribution of [(3)H]NA injected at the lumbar enlargement revealed that at least 97% of the activity recovered from the central nervous system was located in the spinal cord, primarily within the thoracic and upper lumbar segments. Thus, the thermoregulatory effects of intrathecal NA are not likely to be due to an action at supraspinal sites.3. Studies with [(3)H]NA also indicated that significant levels of unmetabolized labelled NA were present in plasma as early as 3 min after intrathecal injection. However, by 15 min after injection, 75% of this radioactivity had disappeared. There was a good temporal correlation between the transient appearance of high levels of NA in plasma and the initial hyperthermic effect of NA. Moreover, the hyperthermia was not inhibited by mecamylamine-induced ganglionic blockade. These results indicate that the initial hyperthermic effect of intrathecal NA is due to a direct action of this monoamine at peripheral sites subsequent to leakage from the spinal subarachnoid space.4. In anaesthetized rats, NA (0.30 mumole) and clonidine (0.035 mumole) injected intrathecally at the lumbar enlargement produced a sustained decrease in neural activity recorded from the lumbar sympathetic chain, a finding which suggests that the vasodilation and hypothermia produced by intrathecal NA are due to an inhibition of sympathetic outflow.5. To investigate the spinal site of action of NA on thermoregulation, rats were prepared with spinal catheters which extended either to the upper cervical region or the lower sacral area. Studies with [(3)H]NA showed that these modifications of the catheter lengths altered the accessability of NA to the intermediolateral nucleus (i.m.l.) of the spinal cord. Injections of NA through the cervical and sacral catheters elicited thermoregulatory effects which differed from those elicited by injections near the lumbar enlargement. The differences were consistent with the hypothesis that the hypothermia and tail skin vasodilation elicited by NA injected at the lumbar enlargement are mediated, at least in part, via a direct inhibitory effect of this monoamine on sympathetic preganglionic neurones located in the i.m.l.6. The effect on mean arterial blood pressure of intrathecal injection of NA (0.30 mumole) at the lumbar enlargement was examined in unanaesthetized rats fitted with chronic arterial catheters. These injections produced an immediate increase in blood pressure. However, this effect was transient and, during most of the time when NA-induced vasodilation and hypothermia were present, blood pressure was normal or only slightly elevated. Thus, it is not likely that a baroreceptor-mediated reflex inhibition of sympathetic outflow contributed significantly to the vasodilatory or hypothermic action of NA.

The material for this study of the spinal cord of Galago consisted of five specimens, and, for comparison, all the segments of a human spinal cord. The spinal cord of Galago is very similar to that of the human fetus, which is not as well defined as in the adult. The lumbar enlargement is as great as the cervical one. The examination of cross-sections reveals a significant reduction in the area of the funiculus posterior, which places this animal between rabbit and bear in this regard. However, its relative percentage graphs are very close to those of the rhesus monkey. A cytoarchitectonic study of Galago shows that its neurons are relatively larger than those in man. Topographic cell distribution in this species is very similar to that of man. The areas of the limbs seem to have no correspondence to other regions of the spinal cord. On the whole, in spite of the specialization of movements of the head and hindquarters, the spinal cord of Galago shows a lesser degree of differentiation.