Muscle Afferents Innervating Skin Form Somatotopically Appropriate Connections in the Adult Rat Dorsal Horn

We have studied the somatotopic reorganization in dorsal horn neurons after a disruption in the normal spatial arrangement of primary sensory axons in adult rats. Muscle afferents were redirected to skin by cutting and cross-anastomosing the hindlimb gastrocnemius nerve (GN) and sural nerve (SN). It has previously been shown that after 10 – 12 weeks GN afferents innervate the hairy skin of the lateral ankle and calf (previously innervated by SN afferents) and become potentially capable of relaying information on the location and intensity of stimuli applied to the skin. We determined the receptive field and response properties of dorsal horn neurons in the lumbar spinal cord, in regions where the lower hindlimb is normally represented. In control animals (with intact or self-anastomosed sural nerves) very few neurons (<8%) received any synaptic input from the GN as assessed by electrical stimulation of the nerve. In contrast, when this nerve innervated skin, many cells responded to GN stimulation, and these nearly all had receptive field components in the former SN territory. Moreover, in animals with cross-anastomosed nerves, cells without GN inputs all had receptive fields outside the former SN skin territory. We have shown that in all likelihood GN afferents substituted for SN afferents in subserving the low and high threshold receptive fields of dorsal horn neurons. Furthermore, for many neurons, receptive fields were formed from inappropriately regrown GN afferents and adjacent intact cutaneous afferents (in the tibial or common peroneal nerves). Therefore, when GN afferents innervate skin in adult animals, they alter their central connectivity in an appropriate manner for their new peripheral terminations, so that an orderly somatotopic representation of the hind limb skin is maintained. We suggest that this plasticity of dorsal horn somatotopy is driven in part by activity-dependent mechanisms.     

Regulation of afferent connectivity in the adult spinal cord by nerve growth factor

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.     

Peripheral specification of sensory neurons transplanted to novel locations along the neuraxis

Thoracic dorsal root ganglia in bullfrogs contain sensory neurons that innervate the skin of the trunk and have synaptic connections in the dorsal horn of the spinal cord. The ganglion that innervates the forelimb contains, in addition to cutaneous afferents, many muscle afferents that project more ventrally in the spinal cord and make monosynaptic connections with motoneurons. In the present study, we have transplanted thoracic sensory neurons to the brachial level in tadpoles to discover whether they can innervate forelimb muscles and, if so, whether they form central connections characteristic of forelimb muscle afferents. The ganglion that normally supplies the forelimb was removed from tadpoles and replaced with 2 thoracic ganglia. After the tadpoles completed metamorphosis, the peripheral and central connections of the transplanted thoracic sensory neurons were examined with anatomical and electrophysiological techniques. When the ganglia were transplanted at stage XIV or earlier, transplanted sensory neurons innervated the forelimb and projected into the brachial spinal cord. Electrical stimulation of forelimb muscle nerves evoked impulses in the dorsal root, indicating that some centrally projecting sensory neurons were muscle afferents. Furthermore, muscle afferents were also activated by stretching muscles which suggest that they terminated on spindles. HRP labeling of the central projections revealed that transplanted sensory neurons terminated at sites characteristic of both cutaneous and muscle afferents. The pattern of synaptic connections was assessed by recording intracellularly from motoneurons. Stimulation of muscle afferents produced monosynaptic EPSPs in motoneurons. As in normal frogs, triceps muscle afferents projected more strongly to triceps motoneurons than to subscapularis and pectoralis motoneurons, while subscapularis afferents projected to all 3 types of motoneurons. Thus, the transplanted sensory neurons formed central connections appropriate to their novel peripheral targets. These observations suggest that interactions between sensory neurons and their targets may be important in determining their central connections.     

Synaptic plasticity in the adult spinal dorsal horn: the appearance of new functional connections following peripheral nerve regeneration

Peripherally regenerated fibers were impaled in the dorsal columns. Each impaled fiber's adequate stimulus was determined and the fiber was activated by passing brief (200 ms) current pulses through the microelectrode. Cord dorsum potentials (CDPs) elicited by fiber stimulation were recorded at 8 sites, and then the fiber was injected with Neurobiotin (NB). In the same preparations, dorsal horn cells were impaled and their receptive fields (RFs) mapped; areas of skin from which the most vigorous responses were elicited were noted. Needle electrodes inserted into these cutaneous "hot spots" were used to electrically activate minimal numbers of peripherally regenerated fibers while simultaneously recording the resulting CDPs and any intracellular EPSPs. This allowed determination of connectivity between regenerated fibers and dorsal horn cells with overlapping RFs. In agreement with findings in intact animals, NB revealed long-ranging collaterals which were not seen using intraaxonally injected horseradish peroxidase (HRP). Although there was no qualitative difference in their morphology compared to those seen in controls, the correlation between spatial distribution of boutons and amplitudes of the monosynaptic CDPs of peripherally regenerated fibers revealed significant shifts in the functional efficacy of many central connections. Transcutaneous electrical stimulation revealed a significantly higher incidence of connectivity between regenerated fibers and cells with overlapping RFs at 9-12 months (86%) than at 5-6 months (34%). Although there was no obvious anatomical reorganization of afferent projections in the dorsal horn, the observed functional changes with time following transection show the formation of new functional central connections.     

The somatotopic organization of primary afferent terminals in the superficial laminae of the dorsal horn of the rat spinal cord

Transganglionic transport of wheatgerm agglutinin conjugated horse-radish peroxidase (WGA-HRP) was used to reveal the central distribution of terminals of primary afferent fibers from peripheral nerves innervating the hind leg of the rat. In separate experiments the sizes and locations of cutaneous peripheral receptive fields were determined by electrophysiological recording techniques for each of the nerves that had been labeled with WGA-HRP. By using digital image analysis, the sizes and positions of the peripheral receptive fields were correlated with the areas of superficial dorsal horn occupied by terminals of primary afferents from each of these receptive fields. Data were obtained from the posterior cutaneous nerve of the thigh, lateral sural, sural, saphenous, superficial peroneal, and tibial nerves. The subdivisions of the sciatic nerve, the sural, lateral sural, superficial peroneal, and tibial nerves each projected to a separate and distinct region of the superficial dorsal horn and collectively formed a "U"-shaped zone of terminal labeling extending from lumbar spinal segments L2 to the caudal portions of L5. The gap in the "U" extended from L2 to the L3-4 boundary and was occupied by terminals from the saphenous nerve. Collectively, all primary afferents supplying the hindlimb occupied the medial 3/4 of the superficial dorsal horn with terminals from the tibial nerve lying most medially and occupying the largest of all the terminal fields. Afferents from the superficial peroneal lay in a zone between the medially situated tibial zone and the more laterally placed sural zone. Afferents from the posterior cutaneous nerve were located most caudally and laterally. Terminal fields from the posterior cutaneous and saphenous nerves differed from the others in having split representations caused presumably by their proximity to the mid-axial line of the limb. Comparisons between the peripheral and the central representations of each nerve revealed that 1 mm2 of surface area of the superficial dorsal horn serves approximately 600-900 mm2 of hairy skin and roughly 300 mm2 of glabrous skin. The vast majority of terminal labeling observed in the dorsal horn was found in the marginal layer and substantia gelatinosa, suggesting that small diameter afferents have an orderly somatotopic arrangement in which each portion of the skin surface is innervated by afferent fibers that terminate in preferred localities within the dorsal horn.     

Regeneration of sensory-motor synapses in the spinal cord of the bullfrog

Sensory fibers innervating muscles in the arm of the bullfrog form specific patterns of monosynaptic connections with motoneurons in the spinal cord. We show here that these normal patterns are re-established after interruption of the second dorsal root (DR2) in tadpoles and postmetamorphic frogs. DR2 was either cut or crushed, and 2 to 8 months later the extent and specificity of regeneration were assessed anatomically and physiologically. Horseradish peroxidase labeling of DR2 showed that sensory afferents had regenerated back into the spinal cord to form local arborizations, as in the normal adult. However, few long-tract fibers in the dorsal columns regenerated. Intracellular recording from different classes of motoneurons at the level of DR2 revealed that triceps muscle sensory afferents had regenerated to form functionally appropriate synapses. As in the normal adult, stimulation of the triceps nerves elicited larger monosynaptic EPSPs in triceps motoneurons than in non-triceps motoneurons. Thus, in the central nervous system of the bullfrog, specific monosynaptic connections are re-formed within the region of local regeneration.     

Difference in central projection of primary afferents innervating facial and intraoral structures in the rat

Transganglionic transport of horseradish peroxidase-wheat germ agglutinin conjugate was used to study the central projection of primary afferent neurons innervating facial and intraoral structures. The examined primary neurons innervating the facial structures were those comprising the frontal and zygomaticofacial nerves and those innervating the cornea, while the primary neurons innervating the intraoral structures included those innervating the mandibular incisor and molar tooth pulps and those comprising the palatine nerve. The primary afferents innervating the facial structures project to the lateral or ventral parts of the trigeminal principal, oral and interpolar subnuclei, and to the rostral cervical spinal dorsal horn across laminae I through V, with a greater proportion being directed to the spinal dorsal horn. The primary afferents innervating the intraoral structures terminate in the dorsomedial subdivisions of the trigeminal principal, oral and interpolar subnuclei, and in laminae I, II, and V of the medial medullary dorsal horn, with a much denser projection being distributed to the rostral subnuclei. In addition to the above brain stem trigeminal sensory nuclear complex, they project to the supratrigeminal nucleus, caudal solitary tract nucleus, and paratrigeminal nucleus. These observations agree with previously reported data that the central projection of trigeminal nerve is organized in different manners for the facial and intraoral structures. Furthermore, the present findings in conjunction with our previous studies clarify that the central projection of primary afferents from the facial skin is organized in a clear somatotopic fashion and that the terminal fields of primary afferents from the intraoral structures extensively overlap in the brain stem trigeminal nuclear complex particularly in its rostral subdivisions. The central mechanism of trigeminal nociception is discussed with particular respect to its difference between the facial and intraoral structures.     

Morphology and somatotopic organization of the central terminals of hindlimb hair follicle afferents in the rat lumbar spinal cord

The morphology of the central collateral arborizations of 24 A-beta hair follicle afferents (HFAs) innervating different regions of the skin of the hindlimb were studied by the intra-axonal injection of horseradish peroxidase (HRP) in adult rats. A total of 236 collaterals were recovered. These fell into three classes--complex, simple, and blind-ending--based on numbers of boutons and terminal branch patterns. The morphology of the HFA central arbors innervating the lateral and medial leg and dorsum of the foot was flame-shaped. Afferents with receptive fields on the glabrous-hairy skin border consistently had extra terminal branches running ventromedially into laminae IV/V. Differences in the width of terminal arbors were found. HFA terminals innervating the lateral leg formed narrower sheets than those innervating the dorsum of the foot and toes. The somatotopic organization of the collaterals and terminal arborizations of individual afferents were analyzed both by considering all the collaterals along an axon's rostrocaudal extent and by only examining arbors with boutons (the complex and simple arbors). Thirty-seven percent of blind-ending and 18% of simple collaterals were found to overlap in the rostrocaudal direction with the complex arborizations of afferents whose receptive fields were in a different cutaneous nerve territory. There was no overlap between complex arborizations of afferents from different nerve territories. However, the complex arbors of afferents with receptive fields within a particular nerve territory showed considerable terminal overlap even if they had nonadjacent peripheral receptive fields. The topographical organization of the central terminals of HFAs, forms a coarse somatotopic map of overlapping terminals whereby a particular region of dorsal horn has a maximal, but not exclusive, input from a particular area of skin.     

Peripheral and central anatomical organization of cutaneous afferent subtypes in a rat nociceptive intersegmental spinal reflex

Stimulation of rat segmental dorsal cutaneous nerves (DCNs) evokes the nociceptive intersegmental cutaneus trunci muscle (CTM) reflex. The reflex consists of early and late responses, mediated by Aδ and C fibers, respectively, based on required stimulation strengths, and shows segmental differences in terms of amplitude and duration. We have now investigated whether the peripheral or central anatomy of nociceptive afferent subtypes in different DCNs also vary in a segmental manner. The numbers of different axon subtypes, determined by axon diameter, were analyzed across peripheral DCNs from T6 to L1. The central projections of T7 and T13 DCN afferents were traced using DCN injections of cholera toxin subunit B (CTB) for myelinated A fibers and isolectin B4 (IB4) for unmyelinated C fibers and both labels were quantified in the dorsal horns. Peripheral axon subtype numbers did not differ significantly across DCNs. Centrally, IB4⁺, but not CTB⁺, projection areas were different between T7 and T13, consistent with different segmental CTM neurogram responses. At both levels, A fibers projected to deeper layers of the dorsal horn than did C fibers. These termination sites are consistent with both mono‐ and polysynaptic connections between these afferents and the ascending propriospinal interneurons of the reflex. Also analyzed were the spatial distribution, the synaptic termination, and the glutamatergic transporter profiles of DCN A and C fibers and their relationship to calcitonin gene‐related peptide (CGRP) staining in the dorsal horn.     

Synaptic activation of the interneurons of the thoracic portion of the spinal cord by reticulospinal fibers of the lateral funiculus

Synaptic processes evoked in various functional groups of thoracic interneurons (Th10,11) by stimulation of ipsi- and contralateral bulbar reticular formation were studied in anesthetized cats with lesions of the spinal cord that remained intact only the ipsilateral funiculus. Activation of reticulospinal fibres of the lateral funiculus with conduction velocities of 30-100 m/s evoked monosynaptic EPSPs in the following types of cells tested: monosynaptically excited by group la muscle afferents; excited by flexor reflex afferents; excited mainly by descending systems; excited by low-threshold cutaneous afferents to a less extent. All these neurons with responses to reticular stimulation were located predominantly in the central and lateral regions of Rexed's lamina VII. Most of the cells in the dorsal horn were not affected by short-latency reticulofugal influences. The only exception were 6 neurons located in the horn most dorsal laminae. Functional organization of connections between the lateral reticulospinal pathways and spinal neurons is discussed as compared to that of medial reticulo-spinal pathways as well as to the organization of "lateral" descending systems: cortico- and rubro-spinal.     

Monosynaptic connections between neurons of trigeminal mesencephalic nucleus and jaw-closing motoneurons in the rat: an intracellular horseradish peroxidase labelling study

In order to confirm the monosynaptic connections of muscle spindle-mediated jaw stretch reflexes, 8 neurons of trigeminal mesencephalic nucleus innervating masseteric muscle spindles were identified electrophysiologically and stained intracellularly with horseradish peroxidase. These axon terminals projected to ipsilateral dorsal and dorsolateral divisions of trigeminal motor nucleus and extensive premotor areas. Under electron microscope, labeled terminals made monosynaptic contacts predominantly with dendrites in the jaw-closing motoneuron pools. One labeled and many non-labeled terminals were frequently observed to converge simultaneously on one dendrite in the area. However, it was of particular interest that 28% of the labeled terminals constituted the intermediate component of axo-axodendritic synaptic triads. The present study confirmed, for the first time, monosynaptic connections between jaw-closing muscle spindle afferents and jaw-closing motoneurons. These findings also provided ultrastructural evidence for the monosynaptic excitation of muscle spindle-mediated jaw stretch reflexes which received presynaptic and postsynaptic inhibitions of the premotor neurons from other sources.     

Tuning of spinal networks to frequency components of spike trains in individual afferents

Cord dorsum potentials (CDPs) evoked by primary afferent fiber stimulation reflect the response of postsynaptic dorsal horn neurons. The properties of these CDPs have been shown to vary in accordance with the type of primary afferent fiber stimulated. The purpose of the present study was to determine the relationships between frequency modulation of the afferent input trains, the amplitude modulation of the evoked CDPs, and the type of primary afferent stimulated. The somata of individual primary afferent fibers were impaled in the L7 dorsal root ganglion of alpha-chloralose-anesthetized cats. Action potentials (APs) were evoked in single identified afferents via the intracellular microelectrode while simultaneously recording the response of dorsal horn neurons as CDPs, or activity of individual target interneurons recorded extracellularly or intracellularly. APs were evoked in afferents using temporal patterns identical to the responses of selected afferents to natural stimulation of their receptive fields. Two such physiologically realistic trains, one recorded from a hair follicle and the other from a slowly adapting type 1 receptor, were chosen as standard test trains. Modulation of CDP amplitude in response to this frequency-modulated afferent activity varied according to the type of peripheral mechanoreceptor innervated. Dorsal horn networks driven by A beta afferents innervating hair follicles, rapidly adapting pad (Krause end bulb), and field receptors seemed "tuned" to amplify the onset of activity in single afferents. Networks driven by afferents innervating down hair follicles and pacinian corpuscles required more high-frequency activity to elicit their peak response. Dorsal horn networks driven by afferents innervating slowly adapting receptors including high-threshold mechanoreceptors exhibited some sensitivity to the instantaneous frequency, but in general they reproduced the activity in the afferent fiber much more faithfully. Responses of synaptically coupled dorsal horn neurons belonging to either hair follicle or SA1 fiber-driven networks to frequency-modulated input were in agreement with the CDP results, confirming that CDP amplitude modulation is a true reflection of EPSP amplitude modulation in at least a subset of dorsal horn neurons comprising the network.     

Plasticity of primary afferent acid phosphatase expression following rerouting of afferents from muscle to skin in the adult rat

We have examined the possibility that reinnervation of a new peripheral target by primary afferent neurones can alter the histochemical properties of those afferents in the adult rat. The hindlimb sural and gastrocnemius nerves largely supply skin and muscle, respectively. In adult animals these nerves were cut and rejoined to either their own distal stumps (self-anastomosis) or that of the other nerve (cross-anastomosis) and allowed to regenerate for 12-16 weeks to reinnervate an appropriate or inappropriate target. Fluoride-resistant acid phosphatase (FRAP) is a chemical marker found in many unmyelinated afferents. We have determined the FRAP expression in normal and regrown nerves and examined its distribution in the dorsal horn of animals with self- and cross-anastomosed nerves. While normal and self-anastomosed sural nerves stained heavily for FRAP, gastrocnemius nerves showed either no staining or only the occasional fibre. Cross-anastomosed gastrocnemius nerves, now innervating the skin, showed a significant increase in staining, in some cases approaching the levels normally seen in sural nerves. Conversely, cross-anastomosed sural nerves (innervating muscle) showed decreased FRAP staining. In the normal dorsal horn the terminals of FRAP containing afferents form a thin band extending throughout the mediolateral extent of lamina II (Devor and Claman: Brain Res. 190:17-28, '80). One week after axotomy of the sural nerve, FRAP is depleted from its terminals and a gap appears in the normal FRAP staining pattern in the lumbar enlargement of the spinal cord. The new expression of FRAP in cross-anastomosed nerves was also seen in their terminals in the dorsal horn.(ABSTRACT TRUNCATED AT 250 WORDS)     

Morphology and somatotopy of the central arborizations of rapidly adapting glabrous skin afferents in the rat lumbar spinal cord

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. © 1993 Wiley-Liss, Inc.     

Organization of cutaneous ventrodorsal and rostrocaudal axial lines in the rat hindlimb and trunk in the dorsal horn of the spinal cord

The somatotopic organization of cutaneous primary afferents projecting to the dorsal horn of the rat spinal cord was investigated. The fluorescent neurotracer, 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) was applied to cutaneous incisions made along ventrodorsal axial lines (VDALs) or rostrocaudal axial lines (RCALs) of the trunk and hindlimb. DiI-induced fluorescent zones appeared in laminae I-III of the dorsal horn in the transverse section. Several fluorescent zones appeared at different mediolateral portions after tracer application to VDALs. After tracer was applied to RCALs, a single zone of fluorescence was observed. Serial transverse sections were used to reconstruct fluorescent zones in lamina II and to illustrate the rostrocaudally elongated band-like projection fields in a horizontal plane. In the horizontal plane, the fluorescent zones of VDALs were reconstructed to band-like projection fields. These fields were arranged mediolaterally and extended rostrocaudally for approximately the length of one spinal cord segment or less. The fluorescent zones of RCALs were reconstructed to one band-like projection field. This field extended rostrocaudally over several spinal cord segments. Cutaneous afferents from the ventral median line of the trunk, tail, hindlimb, sole, and ventral side of the digits projected to the medial margin of the dorsal horn. Cutaneous afferents from the dorsal median lines projected to the lateral margin of the dorsal horn. By analyzing the pattern of the body surface regions and the VDALs and RCALs, the central projection fields of body surface regions could be hypothesized, based on the central projection fields of the individual VDAL and RCAL afferents. Thus, we established a detailed dorsal view map of the central projection fields of cutaneous primary afferents.     

A critical period for the influence of peripheral targets on the central projections of developing sensory neurons

During development, the projections that sensory neurons make within the spinal cord are influenced by the specific targets they contact in the periphery. If sensory ganglia normally supplying principally cutaneous targets are forced to grow into limb muscles, in early stage tadpoles, many sensory neurons within these ganglia innervate limb muscles and subsequently develop spinal projections appropriate for muscle spindle afferents. If the same procedure is performed with adult frogs, however, these novel projections do not form. In this study, we have determined the developmental stages at which this sensitivity to peripheral targets exists. Axons from sensory neurons in thoracic (largely cutaneous) dorsal root ganglia were re-routed into the front leg at various stages through metamorphosis, and the central spinal projections of these re-routed fibers were assessed with HRP labeling. We found that thoracic sensory axons could be made to project to limb muscles throughout development, but that the central projections of these neurons were only appropriate for spindle afferents if the fibers were re-routed before stage XVIII, shortly before metamorphic climax. Because sensory neurons can regenerate specifically into the appropriate spinal laminae even in adult frogs, these results suggest that changes in either the DRG or the arm musculature occur by stage XVII so that DRG neurons cannot respond to novel peripheral targets.     

Dermatomes and the central organization of dermatomes and body surface regions in the spinal cord dorsal horn in rats

Dermatomes and the associated central projection fields were studied with the application of fluorescent neurotracer, 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI), to 21 reference points on rat trunk and hindlimb skin. Segmental distribution and rostrocaudal central level of dorsal root ganglion (DRG) neurons innervating reference points were examined and DiI-induced fluorescent areas were mapped in the horizontal plane through lamina II of the dorsal horn. Segmental levels of DRG neurons innervating reference points were generally identical to the level determined using dye-extravasation methods. However, innervation of the first digit was situated in the L4 dermatome, not the L3 reported previously using those methods. Generally, afferents from a reference point projected to a single field in the ipsilateral dorsal horn. Reference points on ventral and dorsal median lines of the trunk were represented bilaterally. Afferents from reference points located on the ventral median line of the hindlimb projected to two separate fields: one on the medial margin of spinal cord segments L2-L5 and the other on the medial half of spinal cord segment L5. From the distribution of central projection fields of reference points, central projection fields of dermatomes were revealed as even in shape and located within corresponding spinal cord segments. The arrangement of peripheral and central fields of dermatomes and body surface regions suggests that peripheral and central projection fields of cutaneous afferent fibers are reshaped from the common prototypical pattern that exhibits an orderly and evenly sequenced arrangement.     

Organization of hindlimb muscle afferent projections to lumbosacral motoneurons in the chick embryo.

We have examined the organization of muscle afferent projections to motoneurons in the lumbosacral spinal cord of chick embryos between stage 37, when muscle afferents first reach the motor nucleus, and stage 44, which is just before hatching. Connectivity between afferents and motoneurons was assessed by stimulating individual muscle nerves and recording the resulting motoneuron synaptic potentials intracellularly or electrotonically from other muscle nerves. Most of the recordings were made in the presence of DL-2-amino-5-phosphonovaleric acid (APV), picrotoxin, and strychnine to block long-latency excitatory and inhibitory pathways. Activation of muscle afferents evoked slow, positive potentials in muscle nerves but not in cutaneous nerves. These potentials were abolished in 0 mM Ca2+, 2mM Mn2+ solutions, indicating that they were generated by the action of chemical synapses. The muscle nerve recordings revealed a wide-spread pattern of excitatory connections between afferents and motoneurons innervating six different thigh muscles, which were not organized according to synergist-antagonist relationships. This pattern of connectivity was confirmed using intracellular recording from identified motoneurons, which allowed the latency of the responses to be determined. Short-latency potentials in motoneurons were produced by activation of homonymous afferents and the heteronymous afferents innervating the hip flexors sartorius and anterior iliotibialis. Stimulation of anterior iliotibialis afferents also resulted in some short-latency excitatory postsynaptic potentials (EPSPs) in motoneurons innervating the knee extensor femorotibialis, though other connections were of longer latency. Afferents from the adductor, a hip extensor, did not evoke short-latency EPSPs in any of these three types of motoneurons. Short-latency, but not long-latency EPSPs, persisted during repetitive stimulation at 5 Hz, suggesting that they were mediated monosynaptically. Long-latency, fatigue-sensitive potentials were maintained in the presence of APV, picrotoxin, and strychnine, suggesting that polysynaptic pathways utilize non-NMDA receptors as well as NMDA receptors. We found no difference in the pattern of inputs to femorotibialis motoneurons between stage 37-39 and near hatching at stage 44, suggesting muscle afferent projections to these motoneurons are correct at stage 37, when the afferents first reach the lateral motor column in substantial numbers.     

Identity of myelinated cutaneous sensory neurons projecting to nocireceptive laminae following nerve injury in adult mice

It is widely thought that, after peripheral injury, some low‐threshold mechanoreceptive (LTMR) afferents “sprout” into pain‐specific laminae (I–II) of the dorsal horn and are responsible for chronic pain states such as mechanical allodynia. Although recent studies have questioned this hypothesis, they fail to account for a series of compelling results from single‐fiber analyses showing extensive projections from large‐diameter myelinated afferents into nocireceptive layers after nerve injury. Here we show that, in the thoracic spinal cord of naïve adult mouse, all myelinated nociceptors gave rise to terminal projections throughout the superficial dorsal horn laminae (I–II). Most (70%) of these fibers had large‐diameter axons with recurving flame‐shaped central arbors that projected throughout the dorsal horn laminae I–V. This morphology was reminiscent of that attributed to sprouted LTMRs described in previous studies. After peripheral nerve axotomy, we found that LTMR afferents with narrow, uninflected somal action potentials did not sprout into superficial laminae of the dorsal horn. Only myelinated noiceptive afferents with broad, inflected somal action potentials were found to give rise to recurving collaterals and project into superficial “pain‐specific” laminae after axotomy. We conclude that the previously undocumented central morphology of large, myelinated cutaneous nociceptors may very well account for the morphological findings previously thought to require sprouting of LTMRs. J. Comp. Neurol. 508:500–509, 2008. © 2008 Wiley‐Liss, Inc.     

Galanin in sensory neurons in the spinal cord.

The distribution and physiological effects of the neuropeptide galanin (GAL) have been examined in the somatosensory system. GAL is normally present in a few sensory neurons that terminate in the dorsal horn of the spinal cord and it is colocalized with substance P and calcitonin gene-related peptide. After peripheral nerve section, but not dorsal root section, the amount of GAL produced and present in sensory fibers proximal to the section is dramatically upregulated. In parallel functional studies, we could demonstrate that exogenous GAL has a complex effect on the spinal cord reflex excitability, facilitatory at low doses and inhibitory at high doses. Furthermore, GAL inhibits the effect of excitatory neuropeptides physiologically released at the peripheral and central terminals of small diameter afferents that subserve a nociceptive function. After axotomy, the inhibitory effect of GAL is increased. We conclude that GAL may have an important role in the control of nervous impulses that underlie pain states that can occur after peripheral nerve injury.