Nitric oxide synthase-like immunoreactivity in lumbar dorsal root ganglia and spinal cord of rat and monkey and effect of peripheral axotomy

With the immunofluorescence technique, nitric oxide synthase (NOS)-like immunoreactivity (LI) was found in a few medium-sized and small sensory neurons in lumbar (L) 4 and L5 dorsal root ganglia (DRG) of normal rat, and in most of these neurons, NOS-LI coexisted with calcitonin gene-related peptide and sometimes with substance P and galanin. NOS-immunoreactive nerve fibers, terminals and small neurons were also located in the dorsal horn of the segments 4 and 5 of the rat lumbar spinal cord with the highest density in inner lamina II. Many NOS-positive neurons and fibers were seen in the area around the central canal. A sparse network of NOS-immunoreactive nerve fibers was found in the ventral horn. After unilateral sciatic nerve cut in the rat, the number of NOS-positive neurons increased in the ipsilateral L4 and L5 DRGs, mainly in medium and small neurons, but also in some large neurons and very small neurons. NOS-LI could now also be seen in the ipsilateral dorsal roots, and in an increased number of fibers and terminals in both outer and inner lamina II of the ipsilateral dorsal horn. The number of NOS-immunoreactive neurons in lamina II of the ipsilateral dorsal horn was reduced. In the monkey L4 and L5 DRGs, many small neurons were NOS-immunoreactive, but only a few weakly stained nerve fibers and terminals were found in laminae I-IV of the dorsal horn at L4 and L5 lumbar levels. A few NOS-positive neurons were present in lamina X. The number of NOS-immunoreactive neurons was somewhat reduced in DRGs 14 days after peripheral axotomy, but no certain effect was seen in the dorsal horn. These results, together with earlier in situ hybridization studies, demonstrate that axotomy in rat induces a marked upregulation of NOS synthesis in primary sensory neurons, thus suggesting a role for NO in lesioned sensory neurons. In contrast, no such effect was recorded in monkey, perhaps indicating distinct species differences. © 1993 Wiley-Liss, Inc.     

Alterations in the distribution of stimulus-evoked c-fos in the spinal cord after neonatal peripheral nerve injury in the rat

Neonatal peripheral nerve injury results in a significant rearrangement of the central terminals of surviving axotomized and adjacent intact primary afferents in the dorsal horn of the spinal cord. This study investigates the ability of these afferents to make functional contacts with dorsal horn cells, using c-fos expression as a marker of synaptic activation. Graded electrical stimulation at A- or C-fiber strength of either the neonatally axotomized sciatic nerve or the adjacent uninjured saphenous nerve was performed in adult rats. Stimulation of the contralateral uninjured nerve served as a control. Quantitative examination of the number and distribution of c-fos-labeled cells in the spinal cord laminae was performed. Electrical stimulation of the previously axotomized sciatic nerve at A-fiber intensity resulted in many labeled profiles in laminae I-V of the lumbar spinal cord on the experimental as compared to the contralateral side. Electrical stimulation of uninjured saphenous nerve or saphenous-nerve-innervated skin (using pin electrodes) at A-fiber intensity did not evoke c-fos. Stimulation of the saphenous nerve at C-fiber intensity, however, resulted in a significant increase in the number and distribution of c-fos-labeled profiles in laminae I-V on the experimental side as compared to the contralateral control side. The results show that the distribution of c-fos-expressing cells after neonatal nerve injury is compatible with the previously demonstrated distribution of sprouting of primary afferents belonging to an uninjured nerve adjacent to an injured nerve, and that the surviving axotomized afferents are capable of transmitting signals to postsynaptic cells. These findings indicate that Abeta afferent stimulation of injured but not uninjured afferents elicits c-fos expression in postsynaptic cells. This may reflect an injury-induced maintenance of a normal developmental process whereby Abeta stimulation elicits c-fos in dorsal horn neurons.     

Distribution of RET immunoreactivity in the rodent spinal cord and changes after nerve injury

RET (for “rearranged during transfection”) is a transmembrane tyrosine kinase signaling receptor for members of the glial cell line‐derived neurotrophic factor (GDNF) family of ligands. We used RET immunohistochemistry (IHC), double‐labeling immunofluorescence (IF), and in situ hybridization (ISH) in adult naïve and nerve‐injured rats to study the distribution of RET in the spinal cord. In the dorsal horn, strong RET‐immunoreactive (‐ir) fibers were abundant in lamina II‐inner (II_i), although this labeling was preferentially observed after an antigen‐unmasking procedure. After dorsal rhizotomy, RET‐ir fibers in lamina II_i completely disappeared from the dorsal horn, indicating that they were all primary afferents. After peripheral axotomy, RET‐ir in primary afferents decreased in lamina II_i and appeared to increase slightly in laminae III and IV. RET‐ir was also observed in neurons and dendrites throughout the dorsal horn. Some RET‐ir neurons in lamina I had the morphological appearance of nociceptive projection neurons, which was confirmed by the finding that 53% of RET‐ir neurons in lamina I colocalized with neurokinin‐1. GDNF‐ir terminals were in close proximity to RET‐ir neurons in the superficial dorsal horn. In the ventral horn, RET‐ir was strongly expressed by motoneurons, with the strongest staining in small, presumably γ‐motoneurons. Increased RET expression following peripheral axotomy was most pronounced in α‐motoneurons. The expression and regulation pattern of RET in the spinal cord are in line with its involvement in regenerative processes following nerve injury. The presence of RET in dorsal horn neurons, including nociceptive projection neurons, suggests that RET also has a role in signal transduction at the spinal level. This role may include mediating the effects of GDNF released from nociceptive afferent fibers. J. Comp. Neurol. 500:1136–1153, 2007. © 2006 Wiley‐Liss, Inc.     

Enrichment of Glutamate-like Immunoreactivity in Primary Afferent Terminals Throughout the Spinal Cord Dorsal Horn

Although several lines of evidence indicate that glutamate is a neurotransmitter in primary afferent terminals, controversies exist on the proportion and types of such terminals that release glutamate. In the present study quantitative analysis of immunogold labelling was used to assess the presence of glutamate-like immunoreactivity in primary afferent terminals in laminae I – V of the rat spinal cord dorsal horn. Anterograde transport of choleragenoid – horseradish peroxidase from a spinal ganglion and tetramethyl benzidine histochemistry were used to identify primary afferent terminals in laminae I and III – V. Presumed C-fibre terminals in lamina II were identified on morphological criteria (dense sinusoid axon terminals). Primary afferent terminals in all dorsal horn laminae displayed significantly higher levels of glutamate-like immunoreactivity than pleomorphic vesicle-containing profiles in laminae III – IV and large neuronal cell bodies in laminae III – V. The density of gold particles over primary afferent terminals also significantly exceeded the average density of gold particles over laminae II and III – IV. The highest densities of gold particles were present over dense sinusoid axon terminals in lamina II. These findings suggest that glutamate, alone or in combination with other neuroactive compounds, is involved in the transfer of all sensory modalities from primary afferent fibres to dorsal horn neurons.     

Neuropeptide Y and Galanin Binding Sites in Rat and Monkev Lumbar Dorsal Root Ganalia and Spinal Cord and Effect of Peripheral Axotomy

Using monoiodinated peptide YY (PYY) and galanin as radioligands, and neuropeptide Y (NPY) fragments, the distribution of NPY binding sites and its subtypes Y1 and Y2, and of galanin binding sites, was investigated in rat and monkey lumbar (L) 4 and L5 dorsal root ganglia (DRG) and spinal cord before and after a unilateral sciatic nerve cut, ligation or crush. Receptor autoradiography revealed that [¹²⁵I]PYY bound to some DRG neurons and a few nerve fibres in normal rat DRG, and most of these neurons were small. NPY binding sites were observed in laminae I–IV and X of the rat dorsal horn and in the lateral spinal nucleus, with the highest density in laminae 1–11. [¹²⁵I]NPY binding was most strongly attenuated by NPY_13–36, a Y2 agonist, and partially inhibited by [Leu³¹,Pro³⁴]NPY, a Y1 agonist, in both rat DRG and the dorsal horn of the spinal cord. These findings suggest that Y2 receptors are the main NPY receptors in rat DRG and dorsal horn, but also that Y1 receptors exist. After sciatic nerve cut, PYY binding markedly increased in nerve fibres and neurons in DRG, especially in large neuron profiles, and in laminae III-IV of the dorsal horn, as well as in nerve fibres in dorsal roots and the sciatic nerve. Incubation with NPY_13–36 completely abolished PYY binding, which was also reduced by [Leu,³¹ Pro³⁴] NPY. However, the increase in PYY binding seen in laminae I–IV of the ipsilateral dorsal horn after axotomy was not observed after coincubation with [Leu³¹, Pro³⁴] NPY. NPY binding sites were seen in a few neurons in monkey DRG and in laminae I-II, X and IX of the monkey spinal cord. The intensity of PYY binding in laminae I-II of the dorsal horn was decreased after axotomy. Galanin receptor binding sites were not observed in rat DRG, but were observed in the superficial dorsal horn of the spinal cord, mainly in laminae I-II. Axotomy had no effect on galanin binding in rat DRG and dorsal horn. However, galanin receptor binding was observed in many neurons in monkey L4 and L5 DRG and in laminae I–IV and X of monkey L4 and L5 spinal cord, with the highest intensity in laminae I-II. No marked effect of axotomy was observed on the distribution and intensity of galanin binding in monkey DRG or spinal cord. The present results indicate that after axotomy the synthesis of NPY receptors is increased in rat DRG neurons, especially in large neurons, and is transported to the laminae I–IV of the ipsilateral dorsal horn and into the sciatic nerve. No such up-regulation of the NPY receptor occurred in monkey DRG after axotomy. The Y2 receptor seems to be the main NPY receptor in DRG and the dorsal horn of the rat and monkey spinal cord, but Y1 receptors also exist. The increase in NPY binding sites in laminae I–IV of the dorsal horn after axotomy partly represents Y1 receptors. In contrast to the rat, galanin binding sites could be identified in monkey lumbar DRG. No effect of axotomy on the distribution of galanin binding sites in rat or monkey DRG and dorsal horn was detected, suggesting their presence on local dorsal horn neurons (or central afferents).     

Galanin Receptor Binding Sites in Adult Rat Spinal Cord Respond Differentially to Neonatal Capsaicin, Dorsal Rhizotomy and Peripheral Axotomy

The discrete distribution and possible changes in specific [¹²⁵I]galanin binding sites were evaluated in the rat spinal cord following neonatal capsaicin treatment, dorsal rhizotomy and sciatic nerve section. The highest density of [¹²⁵I]galanin binding sites in the normal rat spinal cord was particularly evident in the superficial layers of the dorsal horn whereas moderate to low amounts of labelling were associated with the deeper dorsal horn, areas around the central canal and the ventral horn. Capsaicin-treated rats, compared to littermate controls, showed a significant bilateral increase in [¹²⁵I]galanin binding in the superficial laminae of the dorsal horn. Similarly, unilateral dorsal rhizotomy evoked a significant increase in the density of [¹²⁵I]galanin binding sites in the superficial dorsal horn ipsilateral to surgery. Section of the sciatic nerve, on the other hand, induced a significant depletion in [¹²⁵I]galanin binding in laminae I and II of the ipsilateral dorsal horn. These results, in parallel to those reported for galanin immunoreactivity under similar conditions, suggest that [¹²⁵I]galanin binding sites are preferentially located postsynaptically to the primary afferent fibre terminals in the dorsal horn of the spinal cord. Thus it seems that galanin, at the level of the dorsal spinal cord, regulates the processing of nociceptive information by acting on its own class of specific receptors located postsynaptically to primary sensory terminals.     

Spinal cord afferent systems containing the nerve terminal protein NT75

In the adult spinal cord, immunocytochemical staining for NT75 is concentrated in nerve terminals in the superficial laminae of the dorsal horn. Deeper laminae of the dorsal horn contain moderate immunocytochemical labeling, but the ventral horn is only sparsely stained. The origin of spinal nerve terminals containing NT75 was investigated with lesion techniques, colchicine treatment, and retrograde tracing in combination with immunocytochemical staining. Primary afferent neurons express NT75 immunoreactivity and account for most of the dense staining in the superficial dorsal horn and part of the labeling in the deeper laminae. It was found that corticospinal and virtually all brainstem neurons with descending projections to the spinal cord also express NT75 immunoreactivity, including those terminating in the ventral horn. Colchicine treatment of the spinal cord also resulted in NT75 staining in most, if not all, spinal neurons. It appears that neurons in all three major sources of spinal afferents (primary sensory, descending, and intrinsic systems) can express NT75 immunoreactivity, but that some neurons normally contain higher levels of the protein in their nerve terminals. Previous analysis of developing spinal cord has shown widespread, dense NT75 labeling throughout the spinal gray in the early postnatal period, which later becomes restricted to the adult pattern. These studies support the hypothesis that many spinal pathways express high levels of NT75 immunoreactivity during development, but that only certain pathways maintain high levels in the adult. © 1993 Wiley-Liss, Inc.     

Segmental distribution and central projectionsof renal afferent fibers in the cat studied by transganglionic transport of horseradish peroxidase

The segmental and central distributions of renal nerve afferents in adult cats and kittens were studied by using retrograde and transganglionic transport of horseradish peroxidase (HRP). Transport of HRP from the central cut ends of the left renal nerves labeled afferent axons in the ipsilateral minor splanchnic nerves and sensory perikarya in the dorsal root ganglia from T12 to L4. The majority of labeled cells (85%) were located between L1 and L3. A few neurons in the contralateral dorsal root ganglia were also labeled. Labeled cells were not confined to any particular region within a dorsal root ganglion. Some examples of bifurcation of the peripheral and central processes within the ganglion were noted. A small number of preganglionic neurons, concentrated in the intermediolateral nucleus, were also identified in some experiments. In addition, many sympathetic postganglionic neurons were labeled in the renal nerve ganglia, the superior mesenteric ganglion, and the ipsilateral paravertebral ganglia from T12 to L3 Transganglionic transport of HRP labeled renal afferent projections to the spinal cord of kittens from T1 1 to L6, with the greatest concentrations between Ll and L3. These afferents extended rostrocaudally in Lissauer's tract and sent collaterals into lamina I. In the transverse plane, a major lateral projection and a minor medial projection were observed along the outer and inner margins of the dorsal horn, respectively. From the lateral projection many fibers extended medially in laminae V and VI forming dorsal and ventral bundles around Clarke's nucleus. The dorsal bundle was joined by collaterals from the medial afferent projection and crossed to the contralateral side. The ventral bundle extended into lamina VII along the lateroventral border of Clarke's nucleus. Some afferents in the lateral projection could be followed ventrally into the dorsolateral portion of lamina VII in the vicinity of the intermediolateral nucleus. In the contralateral spinal cord, labeled afferent fibers were mainly seen in laminae V and VI These results provide the first anatomical evidence for sites of central termination of renal afferent axons. Renal inputs to regions (laminae I, V, and VI) containing spinoreticular and spinothajamic tract neurons may be important in the mediation of supraspinal cardiovascular reflexes as well as in the transmission of activity from nociceptors in the kidney. In addition, the identification of a bilateral renal afferent projection in close proximity to the thoracolumbar autonomic nuclei is consistent with the demonstration in physiological experiments of a spinal pathway for the renorenal sympathetic reflexes.     

Vanilloid receptor VR1 is both presynaptic and postsynaptic in the superficial laminae of the rat dorsal horn.

Terminals in the rat spinal cord that express the vanilloid receptor VR1 are from small and medium dorsal root ganglion (DRG) neurons and appear prominent in lamina I and inner lamina II. Because primary afferents from these neurons can be myelinated or unmyelinated and their terminals in these laminae can be of various morphological and functional types, we undertook this study to identify the type(s) of VR1-positive afferent fibers and terminals. In the DRG, many small and medium-sized neurons are immunopositive. Under electron microscopy, dorsal root afferents that are immunopositive for VR1 are predominantly unmyelinated. Large numbers of VR1-positive terminals in lamina I are of the nonglomerular type and may contain dense core vesicles. VR1 immunoreactivity in terminals in lamina I is in good agreement with data on noxious, heat-sensitive neurons in the dorsal horn. Two types of glomerular afferent terminals in lamina II also are immunopositive for VR1. In both laminae, most VR1-positive terminals are distinct from substance P-positive terminals. However, the immunoreactivity in lamina II also is prominent in dendrites that are contacted by primary afferent endings. Because we also observed patchy immunostaining in cell bodies in lamina II, this unexpected result may reflect synthesis of VR1 by neurons in this lamina. However, because dorsal rhizotomy abolishes VR1 staining in both laminae I and II, it is suggested that the expression and intracellular dynamics of VR1 in lamina II neurons are controlled by presynaptic input.     

Chronic peripheral nerve section results in a rearrangement of the central axonal arborizations of axotomized A beta primary afferent neurons in the rat spinal cord

In order to investigate the reorganization of the neuropil of the dorsal horn following peripheral nerve injury, the central terminal arborizations of 35 A beta primary afferent neurons, chronically injured by a cut and ligation of the sural nerve 6–12 weeks previously, were studied by the intra-axonal injection of horseradish peroxidase. Their morphology was compared to 13 intact sural nerve hair follicle afferents. Following axotomy, three kinds of morphological abnormalities were observed in the collateral arbors of the 26 afferents that were hair follicle-like. Atrophy with thin stem axons and reduced terminal branch patterns with few boutons was seen in 5 afferents. Sprouting of bouton-containing terminals into lamina I and IIo was found in 8 afferents. Finally, abnormal arborization patterns in the deeper laminae were observed in 29% of the collateral arbors. Changes included the loss in some arbors of a flame-shaped appearance, which is characteristic of hair follicle afferents, atypical branching patterns and ventrally directed axons producing wider and deeper arbors, compared to normal. Axotomy also caused a disruption of the normal somatotopic organizaiton of sural nerve A beta afferents. This disruption manifested as a variability in the normally mediolaterally restricted terminal sheet, with a consequent loss of the strict somatotopic register in the rostrocaudal direction. Damage to the peripheral axon of A beta primary afferents induces a structural reorganization of their central terminals in the dorsal horn of the spinal cord, which may modify sensory input to the central nervous system.     

Reorganization of central terminals of myelinated primary afferents in the rat dorsal horn following peripheral axotomy

We have investigated the time course and extent to which peripheral nerve lesions cause a morphological reorganization of the central terminals of choleragenoid-horseradish peroxidase (B-HRP)-labelled primary afferent fibers in the mammalian dorsal horn. Choleragenoid horseradish peroxidase is retrogradely transported by myelinated (A) sensory axons to laminae I, III, IV and V of the normal dorsal horn of the spinal cord, leaving lamina II unlabelled. We previously showed that peripheral axotomy results in the sprouting of numerous B-HRP labelled large myelinated sensory axons into lamina II. We show here that this spread of B-HRP-labelled axons into lamina II is detectable at 1 week, maximal by 2 weeks and persists for over 6 months postlesion. By 9 months, however, B-HRP fibers no longer appear in lamina II. The sprouting into lamina II occurs whether regeneration is allowed (crush) or prevented (section with ligation), and does not reverse at times when peripheral fibers reinnervate the periphery. We also show that 15 times more synaptic terminals in lamina II are labelled by B-HRP 2 weeks after axotomy than in the normal. We interpret this as indicating that the sprouting fibers are making synaptic contacts with postsynaptic targets. This implies that A-fiber terminal reorganization is a prominent and long-lasting but not permanent feature of peripheral axotomy. We also provide evidence that this sprouting is the consequence of a combination of an atrophic loss of central synaptic terminals and the conditioning of the sensory neurons by peripheral axotomy. The sprouting of large sensory fibers into the spinal territory where postsynaptic targets usually receive only small afferent fiber input may bear on the intractable touch-evoked pain that can follow nerve injury. © 1995 Wiley-Liss, Inc.     

Light microscopic and ultrastructural analysis of GABA-immunoreactive profiles in the monkey spinal cord

It is hypothesized that terminals containing gamma-aminobutyric acid (GABA) participate in presynaptic inhibition of primary afferents. To date, few convincing GABA-immunoreactive (GABA-IR) axo-axonic synapses have been demonstrated in support of this theory. The goal of this study is to document the relationship between GABA-IR profiles and central terminals in glomerular complexes in lumbar cord of the monkey (Macaca fascicularis). In addition, the relationship between GABA-IR profiles and other neural elements are analyzed in order to better understand the processing of sensory input in the spinal cord. GABA-IR cell bodies were present in Lissauer's tract (LT) and in all laminae in the spinal gray matter except lamina IX. GABA-IR fibers and terminals were heavily concentrated in LT; laminae I, II, and III; and present in moderate concentration in the deeper laminae of the dorsal horn, ventral horn (especially in association with presumed motor neurons), and lamina X. Electron microscopic analysis confined to LT and laminae I, II, and III demonstrated GABA-IR cell bodies, dendrites, and myelinated and unmyelinated fibers. GABA-IR cell bodies received sparse synaptic input, some of which was immunoreactive for GABA. The majority of the synaptic input to GABA-IR neurons occurred at the dendritic level. Furthermore, the presence of numerous vesicle-containing GABA-IR dendrites making synaptic interactions indicated that GABA-IR dendrites also provided a major site of output. Two consistent arrangements were observed in laminae I-III concerning vesicle-containing GABA-IR dendrites: 1) they were often postsynaptic to central terminals and 2) they participated in reciprocal synapses. The majority of GABA-IR axon terminals observed contained round clear vesicles and varying numbers of dense core vesicles. Only on rare occasions were GABA-IR terminals with flattened vesicles observed. GABA-IR terminals were not observed as presynaptic elements in axo-axonic synapses; however, on some occasions, GABA-IR profiles presumed to be axon terminals were observed postsynaptic to large glomerular type terminals. Our findings suggest that a frequent synaptic arrangement exists in which primary afferent terminals relay sensory information into a GABAergic system for further processing. Furthermore, GABA-IR dendrites appear to be the major source of input and output for this inhibitory system. The implications of this GABAergic neurocircuitry are discussed in relation to the processing of sensory input in the superficial dorsal horn and in terms of mechanisms of primary afferent depolarization (PAD).     

Distribution of somatic and visceral primary afferent fibres within the thoracic spinal cord of the cat

Transport of horseradish peroxidase (HRP) through somatic and visceral nerve fibres was used to study the patterns of termination of somatic and visceral primary afferent fibres within the lower thoracic segments of the cat's spinal cord. A concentrated solution of HRP was applied for at least 5 hours to the central end of the righ greater splanchnic nerve and of the left T9 intercostal nerve of adult cats. Some animals remained under chloralose anaesthesia for the duration of the HRP transport times (up to 53 hours) whereas longer HRP application and transport times (4-5 days) were allowed in animals that recovered from barbiturate anaesthesia. Somatic afferent fibres and varicosities (presumed terminals) were found in laminae I, II, III, IV, and V of the ipsilateral dorsal horn and in the ipsilateral Clarke's column. The density of the somatic projection was particularly high in the superficial dorsal horn. In parasagittal sections of the cord, bundles of somatic fibres were seen joining the dorsal horn from the dorsal roots via the dorsal columns and Lissauer's tract. A medio-lateral somatotopic arrangement of somatic afferent terminations was observed, with afferent fibres from the ventral parts of the dermatome ending in the medial dorsal horn and afferent fibres from the dorsal parts of the dermatome ending in the lateral dorsal horn. The total rostro-caudal extent of the somatic projection through a single spinal nerve was found to be of 2 and 2/3 segments, including the segment of entry, the entire segment rostral to it and two-thirds of the segment caudal to it. A lateral to medial shift in the position of the somatic projection was observed in the rostro-caudal axis of the cord. Visceral afferent fibres and varicosities (presumed terminals) were seen in laminae I and V of the ipsilateral dorsal horn. The density of the visceral projection to the dorsal horn was substantially lower than that of the somatic projection. Visceral afferent fibres reached the dorsal horn via Lissauer's tract and joined a lateral bundle of fine fibres that run along the lateral edge of the dorsal horn. The substantia gelatinosa (lamina II) appeared free of visceral afferent fibres. These results are discussed in relation to the mechanisms of viscero-somatic convergence onto sensory pathways in the thoracic spinal cord.     

Hyperpolarization-activated and cyclic nucleotide-gated cation channel subunit 2 ion channels modulate synaptic transmission from nociceptive primary afferents containing substance P to secondary sensory neurons in laminae I-IIo of the rodent spinal dorsal horn

We have previously demonstrated that hyperpolarization-activated and cyclic nucleotide-gated cation channel subunit 2 (HCN2) is expressed by terminals of peptidergic nociceptive primary afferents in laminae I-IIo of the rat spinal dorsal horn. In this study, we investigated the possible neurotransmitters and postsynaptic targets of these HCN2-expressing primary afferent terminals in the superficial spinal dorsal horn by using immunocytochemical methods. We demonstrated that HCN2 widely colocalizes with substance P (SP), and that HCN2-positive terminals that are also immunoreactive for SP form serial close appositions with dendrites and perikarya of neurokinin 1 receptor-immunoreactive neurons. It was also found that HCN2-immunoreactive terminals are frequently apposed to neurons that are immunoreactive for calbindin, micro-opioid receptor and the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate receptor subunit GluR2, markers for excitatory interneurons. Investigating HCN2 immunoreactivity in glutamic acid decarboxylase 65-green fluorescent protein transgenic mice, we found that HCN2-positive terminals occasionally also contact cells that contain an isoform of glutamic acid decarboxylase (glutamic acid decarboxylase 65), a marker for GABAergic inhibitory neurons. Application of ZD7288, an antagonist of HCN channels, onto neurons that were recorded in spinal cord slices with whole-cell patch-clamp electrodes reduced the number of monosynaptic excitatory postsynaptic potentials evoked by electrical stimulation of primary afferents at nociceptive intensities. The results suggest that HCN2 may contribute to the modulation of membrane excitability of SP-containing nociceptive primary afferent terminals, may increase the reliability of synaptic transmission from primary afferents to secondary sensory neurons and thus may play a role in the fine-tuning of pain transmission from nociceptive primary afferents to neurons in the spinal dorsal horn.     

Regulation of neuronal and glial galectin-1 expression by peripheral and central axotomy of rat primary afferent neurons

Galectin-1 (Gal1) is an endogenously-expressed protein important for the embryonic development of the full complement of primary sensory neurons and their synaptic connections in the spinal cord. Gal1 also promotes axonal regeneration following peripheral nerve injury, but the regulation of Gal1 by axotomy in primary afferent neurons has not yet been examined. Here, we show by immunohistochemistry and in situ hybridization that Gal1 expression is differentially regulated by peripheral nerve injury and by dorsal rhizotomy. Following peripheral nerve injury, the proportion of Gal1-positive DRG neurons was increased. An increase in the proportion of large-diameter DRG neurons immunopositive for Gal1 was paralleled by an increase in the depth of immunoreactivity in the dorsal horn, where Gal1-positive terminals are normally restricted to laminae I and II. Dorsal rhizotomy did not affect the proportions of neurons containing Gal1 mRNA or protein, but did deplete the ipsilateral dorsal horn of Gal1 immunoreactivity, indicating that it is transported centrally by dorsal root axons. Dorsal rhizotomy also resulted in an increase in Gal1 mRNA the nerve peripheral to the PNS-CNS interface (likely within Schwann cells and/or macrophages), and to a lesser extent within deafferented spinal cord regions undergoing Wallerian degeneration. This latter increase was notable in the dorsal columns and along the prior trajectories of myelinated afferents into the deeper dorsal horn. These results show that neuronal and glial expressions of Gal1 are tightly correlated with regenerative success. Thus, the differential expression pattern of Gal1 following peripheral axotomy and dorsal rhizotomy suggests that endogenous Gal1 may be a factor important to the regenerative response of injured axons.     

Preferential immunohistochemical localization of vasoactive intestinal polypeptide (VIP) in the sacral spinal cord of the cat: light and electron microscopic observations

In the present study we have employed immunoperoxidase techniques to investigate the distribution of vasoactive intestinal polypeptide (VIP)-like immunoreactivity in the spinal cord and sensory ganglia of the cat. The spinal distribution of VIP-containing neuronal processes was also compared with that of substance P (SP), somatostatin (SOM), and cholecystokinin-8 (CCK) at lumbar, sacral, and coccygeal levels. At sacral levels, VIP was found to be contained in small and medium-sized primary sensory neurons and in dorsal rootlets. Deafferentation, by either ganglionectomy or dorsal rhizotomy, resulted in a nearly complete loss of VIP immunoreactivity in the spinal cord. The spinal distribution of VIP fibers and terminals was most dense and extensive in sacral segments. Forming a thin shell around the dorsal horn, collaterals, apparently originating from Lissauer's tract, projected either medially or laterally through lamina I. Laterally, many VIP axons terminated in lateral laminae V to VII. Others projected further through the neck of the dorsal horn to medial lamina V and the gray matter near the central canal. Medially, VIP axons descended through lamina I to expand into terminal fields in the posterior commissure and medial lamina V. At the ultrastructural level, VIP-like immunoreactivity was found in dense core vesicles within axonal enlargements containing both large dense core and smaller clear round vesicles. Synaptic connections were infrequently observed but, when encountered, were of the simple axodendritic type. The spinal distribution of VIP-containing fibers was remarkably similar to that reported for pelvic nerve visceral afferents, both in termination patterns within the spinal gray matter and in localization to the sacral cord. The density of SP-, SOM-, and CCK-containing fibers and terminals was constant at all levels examined (L4 to Co4). In marked contrast, the distribution of VIP fibers, much like that of pelvic nerve afferents, was mostly confined to sacral segments. Thus, although SP, SOM, and CCK may be contained within a population of sacral visceral afferents, they must be common to afferent systems in other segments as well. VIP, however, appears to be preferentially contained within pelvic visceral afferent fibers confined mostly to sacral segments.     

A-fiber sensory input induces neuronal cell death in the dorsal horn of the adult rat spinal cord.

Excitotoxicity due to excessive synaptic glutamate release is featured in many neurological conditions in which neuronal death occurs. Whether activation of primary sensory pathways can ever produce sufficient over-activity in secondary sensory neurons in the dorsal horn of the spinal cord to induce cell death, however, has not been determined. In this study, we asked whether activity in myelinated afferents (A fibers), which use glutamate as a transmitter, can induce cell death in the dorsal horn. Using stereological estimates of neuron numbers from electron microscopic sections, we found that stimulation of A-fibers in an intact sciatic nerve at 10 Hz, 20 Hz, and 50 Hz in 10-minute intervals at a stimulus strength that activates both Abeta and Adelta fibers resulted in the loss of 25% of neurons in lamina III, the major site of termination of large Abeta fibers, but not in lamina I, where Adelta fibers terminate. Furthermore, sciatic nerve lesions did not result in detectable neuron loss, but activation of A fibers in a previously sectioned sciatic nerve did cause substantial cell death not only in lamina III but also in laminae I and II. The expansion of the territory of A-fiber afferent-evoked cell death is likely to reflect the sprouting of the fibers into these laminae after peripheral nerve injury. The data show, therefore, that primary afferent A-fiber activity can cause neuronal cell death in the dorsal horn with an anatomical distribution that depends on whether intact or injured fibers are activated. Stimulation-induced cell death potentially may contribute to the development of persistent pain.     

Fine afferent fibers from viscera do not terminate in the substantia gelatinosa of the thoracic spinal cord

Transganglionic transport of horseradish peroxidase (HRP) has been used to study the anatomy of the central projection of somatic and visceral afferent fibers to the thoracic spinal cord of the cat. A dense concentration of somatic afferent fibers and terminals was found in laminae I and II of the dorsal horn and more scattered terminals were present in laminae III, IV and V and in Clarke's column. In contrast, visceral afferent fibers and terminals were found only in lamina I or reaching lamina V via a small bundle of fibers located in the lateral border of the dorsal horn. These results indicate that fine afferent fibers from viscera, unlike those of cutaneous origin, do not project to the substantia gelatinosa (lamina II) of the dorsal horn.