Reappearance of calcitonin gene-related peptide-like immunoreactivity in the dorsal horn in the long-term dorsal root transected rat.

Calcitonin gene-related peptide (CGRP)-immunoreactive (IR) fibers in the rat dorsal horn superficial laminae vanish almost completely 3 weeks following unilateral dorsal rhizotomy. After a prolonged survival (20 weeks) of dorsal rhizotomy there is, however, a reappearance of CGRP-IR fibers in the corresponding laminae of the dorsal horn. The density of such IR fibres showed a clear gradient with the lowest number found in the midlesion region and an increase in density towards the neighboring, intact segments. In normal as well as lesioned rats, no neurons intrinsic to the dorsal horn contained detectable levels of CGRP-like immunoreactivity (LI). Furthermore, no cells could, by use of in situ hybridization, be demonstrated to contain detectable levels of mRNA encoding for CGRP in the dorsal horn. Based on these findings, we suggest that the CGRP-IR fibers observed following long-term survival of dorsal rhizotomy derive from proliferating collateral branches of primary afferents of neighboring intact segments.     

Increased calcitonin gene-related peptide- and cholecystokinin-like immunoreactivities in spinal motoneurones after dorsal rhizotomy

Summary Possible changes in neuropeptides within the ventral horn of the spinal cord were investigated after unilateral dorsal rhizotomy at the lumbar level (L1–L6) in adult rats. Ten days after the surgery, immunohistochemical observations and radioimmunological determinations confirmed a marked loss of calcitonin gene-related peptide (CGRP)- and substance P (SP)-like immunoreactivities within the superficial layers of the deafferented dorsal horn, as expected from the degeneration of primary afferent fibres containing these peptides. A concomitant increase in immunohistochemical staining and levels of CGRP (+296%) and CCK (+71%)- like immunoreactivities was observed in the ipsilateral ventral horn where both peptides are located in motoneurones. In contrast, substance P-like immunoreactivity that is confined to fibres and terminals within the ventral horn, was not altered by dorsal rhizotomy. These data indicate that the expression of neuropeptides in spinal motoneurones can be influenced by primary afferent inputs.     

Parvalbumin- and calbindin D28k-immunoreactive neurons in the superficial layers of the spinal cord dorsal horn of rat

Immunohistochemical techniques were utilized to investigate the distribution and morphology of neurons containing the calcium binding proteins parvalbumin (PV) and calbindin D28k (CaBP) in the superficial layers of rat spinal cord. Most PV-immunoreactive (PV-IR) neurons were restricted to a 25 to 60 microns thick band straddling the border between lamina II and III. Positive somata had long rostrocaudally oriented dendrites confined to narrow sagittally arranged sheets within this band and axons that entered lamina II or the superficial portions of lamina III. Long varicose axons, presumed to originate from these cells, were moderately distributed in Lissauer's tract and lamina II. CaBP-immunoreactive (CaBP-IR) neurons were found within lamina I and throughout lamina II. Large calibre PV-IR and CaBP-IR axons were seen in the dorsal column and the lateral funiculus. Dorsal rhizotomy or neonatal capsaicin treatment appeared to have no effect on PV-IR and CaBP-IR elements in the superficial lumbar dorsal horn. However, dorsal rhizotomy reduced the number of positive axons in the dorsal column and in deeper lamina of the dorsal horn. These results add to the known lamination patterns of the superficial dorsal horn and point to the existence of a lamina defined by PV-positive neurons at the lamina II/III border. These neurons may have electrophysiological characteristics attributed to PV- or CaBP-containing neurons elsewhere in the CNS.     

NPY Y1 receptors are present in axonal processes of DRG neurons

Using a sensitive immunohistochemical method, the localization of the neuropeptide Y (NPY) Y1 receptor (Y1R) was studied in contralateral and ipsilateral dorsal root ganglion (DRG) neurons of rats subjected to different unilateral manipulations with focus on their axonal processes and projection areas. Y1R-like immunoreactivity (LI) was observed in the contralateral sciatic nerve and dorsal roots of lesioned rats, and double staining revealed colocalization with calcitonin gene-related peptide (CGRP). Y1R-LI was also seen in fibers close to and even within the epidermis. A fairly small number of nerve endings double-labeled for Y1R and CGRP were present in the dorsal horn. After unilateral crush of the sciatic nerve Y1R- and CGRP-LI accumulated in the same axons proximal to the lesion. After dorsal rhizotomy CGRP-LI was strongly reduced in the ipsilateral dorsal horn. No certain change was observed for Y1R- or NPY-LI, but Y1R/CGRP double-labeled nerve endings disappeared after the lesion. These results strongly suggest centrifugal transport of Y1Rs in DRG neurons, mainly to the peripheral sensory branches. To what extent these Y1Rs are functional has not been analyzed here, but a recent study on Y1R null mice provides evidence for involvement of prejunctional Y1Rs in peripheral sensory functions     

Restoration of substance P and calcitonin gene-related peptide in dorsal root ganglia and dorsal horn after neonatal sciatic nerve lesion

Dorsal root ganglion (DRG) neurons decrease their substance P (SP) synthesis after peripheral nerve lesions. Levels in the dorsal horn also decline but return to normal if regeneration is successful. In adults, when regeneration is prevented, recovery of SP in the dorsal horn is slow and incomplete, whereas in newborns, recovery is rapid and complete even though retrograde cell death of DRG neurons is greater than in adults. We have examined the mechanisms that might account for the rapid and complete recovery of SP and calcitonin-gene related peptide (CGRP) in the dorsal horn after peripheral nerve injury in newborns. Peptides were compared in the L4 and L5 DRG and spinal cord segments of normal rats and in rats surviving 6 days to 4 months after sciatic nerve section/ligation within 24 hours of birth. Sciatic nerve section/ligation produced 50% neuron death in L4 and L5 DRGs, but immunocytochemical methods showed that both SP-immunoreactivity (-IR) and CGRP-IR recovered completely in dorsal horn. Radioimmunoassay confirmed that recovery of SP was not an artefact due to shrinkage. β-Preprotachykinin (PPT)-mRNA hybridization and SP-IR were observed mostly in small neurons; α-CGRP-mRNA-hybridized and CGRP-IR neurons were more heterogeneous. The percentage of DRG neurons that contained SP (～ 25%) or CGRP (～ 50%) was the same in normal newborn and adult rats. Neither selective cell survival nor change in neuron phenotype was likely to contribute to the recovery seen in the dorsal horn, and DRG neurons ipsilateral to the lesion exhibited the same level of hybridized β-PPT-mRNA and α-CGRP-mRNA as intact DRG neurons. Because neither the constitutive level of expression of the genes nor peptide levels increased above those observed in intact DRG neurons, these mechanisms were also not responsible. Axotomized DRG neurons, however, contributed to recovery. Recovery was also due to sprouting by neurons in intact DRGs rostral and caudal to L4 and L5. © 1993 Wiley-Liss, Inc.     

The location of GABAB receptor binding sites in mammalian spinal cord

GABAB binding sites in rat spinal cord have been detected by receptor autoradiography using 3H-GABA in the presence of isoguvacine. The sites could be demonstrated throughout the spinal cord grey matter. The maximum concentration of GABAB sites occurred in lamina II with substantial amounts in other laminae of the dorsal horn. Much lower levels were detected in the ventral horn. Unilateral rhizotomy reduced the number of GABAB sites in the dorsal horn without affecting levels in the ventral horn. The greatest reduction occurred in lamina II with 18% loss 2 days after surgery, 23% after 4 days, 25% after 6 days, and 48% after 15 days. The change after 15 days was comparable to that produced 4 months after neonatal capsaicin administration (50 mg/kg). The only apparent difference between rhizotomy and capsaicin treatment occurred in lamina IV, where rhizotomy produced a greater reduction than capsaicin. 3H-Neurotensin binding in sections from the same animals was unaltered after rhizotomy, indicating a lack of change in the populations of neurons containing neurotensin-binding sites. This would support the view that up to 50% of GABAB binding sites are located on nerve terminals. The greater reduction in lamina IV after rhizotomy would suggest that GABAB sites may be present on large-diameter afferent fibres that terminate in this region as well as on smaller-diameter C and A delta fibres.     

Analysis of calcitonin gene-related peptide-like immunoreactivity in the cat dorsal spinal cord and dorsal root ganglia provide evidence for a multisegmental projection of nociceptive C-fiber primary afferents.

Recent studies have suggested that calcitonin gene-related peptide (CGRP) can be used as a marker for a subpopulation of nociceptive primary afferents. Consequently, CGRP-immunoreactive (CGRP-IR) primary afferents have been reported to project many segments rostral to their segment of entry and to send collaterals into the superficial and deep laminae of the dorsal horn. This study reports that some CGRP-IR primary afferents of sacral origin project rostral through the ipsilateral lumbar enlargement in the cat. The ultrastructure of these multisegmentally projecting primary afferent axons and terminals identified in a partially denervated cat was examined and compared to the ultrastructure of CGRP-IR afferents from an intact cat. Retrograde transport of wheatgerm agglutinin-colloidal gold injected into the cat L4 spinal cord resulted in labeling of primary afferent cell bodies in the ipsilateral L4-S1 dorsal root ganglia (DRG). Analysis of every fourth section through the ipsilateral S1 DRG revealed as many as 1,000 retrogradely labeled neuronal cell bodies. One third of these cell bodies were double labeled for CGRP-like immunoreactivity. The number of single- and double-labeled cells increased in ganglia closer to the injection site (L4-L7). At the ultrastructural level, in the lumbosacral dorsal spinal cord of a normal cat, most CGRP-IR axons were unmyelinated, while the rest were small myelinated axons. In both the superficial dorsal horn and lamina V, CGRP-IR varicosities were dome shaped, scallop shaped, or elongated. The CGRP-IR varicosities contained small agranular vesicles and frequently a few dense core vesicles. These labeled varicosities formed asymmetric synapses on unlabeled dendritic spines, shafts, or neuronal somata. One cat received multiple unilateral dorsal rhizotomies (S1-L4) and an ipsilateral hemisection (mid L4). CGRP-IR axons and terminals were found within each of the rhizotomized segments, although their density was greatly reduced compared to that in the intact animals. In Lissauer's tract the majority (greater than 90%) of CGRP-IR fibers were unmyelinated. In laminae I and V, the remaining CGRP-IR varicosities were mainly the dome-shaped varicosities morphologically similar to those observed in the normal spinal cords. They contained small agranular vesicles and a few dense core vesicles and formed asymmetric synapses on unlabeled dendritic shafts and spines. These data demonstrate that unmyelinated, presumably C-fiber nociceptive primary afferents and some small myelinated A-delta nociceptive primary afferents of sacral origin project rostral through the cat lumbar enlargement and make synaptic connections in both the superficial and deep laminae of the cat dorsal spinal cord.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Calcitonin gene-related peptide immunoreactivity in the spinal cord of man and of eight other species

Calcitonin gene-related peptide (CGRP) immunoreactivity was found throughout the entire spinal cord of man, marmoset, horse, pig, cat, guinea pig, mouse, rat, and frog. CGRP-immunoreactive fibers were most concentrated in the dorsal horn. In the ventral horn of some species large immunoreactive cells, tentatively characterized as motoneurons, were present. Pretreatment of rats with colchicine enhanced staining of these large cells but did not reveal CGRP-immunoreactive cell bodies in the dorsal horn. In the dorsal root ganglia, CGRP immunoreactivity was observed in most of the small and some of the intermediate sized cells. Substance P immunoreactivity, where present, was co-localized with CGRP to a proportion of the small cells. In the cat the ratio of substance P-immunoreactive to CGRP-immunoreactive ganglion cells was 1:2.7 (p less than 0.001). The concentration of CGRP-immunoreactive material in tissue extracts was determined by radioimmunoassay. In the dorsal horn of the rat spinal cord the levels of peptide were found to range from 225.7 +/- 30.0 pmol/gm of wet weight in the cervical region to 340.6 +/- 74.6 pmol/gm in the sacral spinal cord. In the rat ventral spinal cord, levels of 15.7 +/- 2.7 to 35.1 +/- 10.6 pmol/gm were found. The concentration in dorsal root ganglia of the lumbar region was 225.4 +/- 46.9 pmol/gm. Gel permeation chromatography of this extractable CGRP-like immunoreactivity revealed three distinct immunoreactive peaks, one eluting at the position of synthetic CGRP and the others, of smaller size, eluting later. In cats and rats, rhizotomy induced a marked loss of CGRP-immunoreactive fibers from the dorsal horn of the spinal cord. In the cat, unilateral lumbosacral dorsal rhizotomy resulted in a significant (p less than 0.05) reduction of extractable CGRP from the ipsilateral lumbar dorsal horn (5.6 +/- 1.2 pmol/gm of wet weight) compared to the contralateral side (105.0 +/- 36.0 pmol/gm of wet weight). We conclude that the major origin of CGRP in the dorsal spinal cord is extrinsic, from afferent fibers which are probably derived from cells in the dorsal root ganglia. The selective distribution of CGRP throughout sensory, motor, and autonomic areas of the spinal cord suggests many putative roles for this novel peptide.     

Large Calibre Primary Afferent Neurons Projecting to the Gracile Nucleus Express Neuropeptide Y after Sciatic Nerve Lesions: an Immunohistochemical and In Situ Hybridization Study in Rats

Using immunohistochemistry and in situ hybridization, we studied changes in expression of some neuropeptides in large and medium-sized neurons in lumbar 4 and 5 rat dorsal root ganglia projecting to the gracile nucleus, in response to peripheral axotomy. Fourteen days after unilateral sciatic nerve transection, many large neurons and some medium-sized neurons in ipsilateral dorsal root ganglia were strongly neuropeptide Y-positive. Galanin-, vasoactive intestinal polypeptide (VIP)- and peptide histidine-isoleucine (PHI)-like immunoreactivities coexisted with neuropeptide Y-like immunoreactivity in some of these neurons. After axotomy numerous large and medium-sized cells contained neuropeptide Y mRNA in the ipsilateral ganglia, whereas no hybridization was seen in the contralateral or control ganglia. Cross-sectioned, large neuropeptide Y-positive fibres were observed in a somatotopically appropriate zone within the ipsilateral gracile fasciculus. A dense network of neuropeptide Y-immunoreactive, large nerve fibres and terminals was seen in the ipsilateral gracile nucleus. A small number of galanin- and VIP/PHI-like immunoreactive nerve fibres and terminals were also observed in adjacent sections. Neuropeptide Y-like immunoreactivity colocalized with galanin- or VIP/PHI-like immunoreactivity in some nerve fibres. None of these neuropeptide immunoreactivities could be detected in nerve fibres and terminals in the control or contralateral gracile nucleus. These findings suggest that neuropeptides, in addition to their role in small dorsal root ganglion neurons, may have a function in large and medium-sized dorsal root ganglion neurons projecting to laminae III and IV in the dorsal horn as well as to the gracile nuclei, as a part of their response to peripheral axotomy.     

A quantitative study of the coexistence of peptides in varicosities within the superficial laminae of the dorsal horn of the rat spinal cord

While several peptides have been shown to coexist in perikarya within dorsal root ganglia of rat, coexistence of peptides has not been confirmed in axons associated with these neurons. In this study, the coexistence of substance P (SP) with somatostatin (SOM), calcitonin gene-related peptide (CGRP), dynorphin A 1-8 (DYN), neurotensin (NT), galanin (GAL), and 5-HT in varicosities was visualized using fluorescence immunohistochemistry. Densities of immunoreactive varicosities within laminae I and II of the dorsal horn of the rat spinal cord were quantified by computer-assisted image analysis. Decreases in densities of immunoreactive varicosities as a result of multiple unilateral dorsal rhizotomies were used to determine proportions of immunoreactive varicosities associated with primary afferent neurons. Three observations were made. (1) Dorsal rhizotomy depleted greater than one-third of the varicosities individually immunoreactive for SP, SOM, GAL, or DYN, confirming the association of these peptides with primary afferent neurons. (2) SP coexisted with CGRP, GAL, and DYN in varicosities within the dorsal horn of normal animals. (3) CGRP-, SP+CGRP-, and SP+GAL-immunoreactive varicosities were nearly depleted following dorsal rhizotomy. The depletion of these peptides, particularly in combination, indicates that they may be used as markers for varicosities of some primary afferent neurons within the superficial laminae of the dorsal horn of the rat spinal cord.     

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.     

Quantitative autoradiographic localization of [125I]neuropeptide Y receptor binding sites in rat spinal cord and the effects of neonatal capsaicin, dorsal rhizotomy and peripheral axotomy.

Using in vitro quantitative receptor autoradiography the present study reports on the distribution and possible changes of [125I]neuropeptide Y (NPY) binding sites in the rat spinal cord following neonatal capsaicin treatment, dorsal rhizotomy and sciatic nerve section. In control spinal cord the highest density of [125I]NPY binding sites was noticed in the superficial layers of the dorsal horn whereas low-to-moderate densities of [125I]NPY binding sites were detected in the deeper dorsal horn and in the ventral horn. In comparison with control rats, neonatally treated capsaicin rats showed a significant (P less than 0.001) bilateral decrease in [125I]NPY binding sites in the superficial layers of the dorsal horn. Unilateral dorsal rhizotomy and unilateral sciatic nerve section also exhibited a significant (P less than 0.05) depletion in [125I]NPY labeling in the superficial layers of the dorsal horn ipsilateral to the surgery. These results suggest that a certain proportion of [125I]NPY receptor sites is located on the primary afferent fibers of the superficial layers of the dorsal horn. This peptide thus could play an important role in the modulation of nociceptive transmission by acting directly on primary afferent terminals.     

Ectopic Substance P and Calcitonin Gene-related Peptide Immunoreactive Fibres in the Spinal Cord of Transgenic Mice Over-expressing Nerve Growth Factor

The aim of this study was to investigate the in vivo effects of CNS over-expression of nerve growth factor (NGF) on primary sensory neurons. To achieve this objective a transgenic mouse model was generated which bore a chick NGF gene driven by the myelin basic protein promoter. Northern blot analysis demonstrated that high levels of NGF mRNA were detected in the spinal cord of adult transgenic mice. Using immunocytochemistry NGF-immunoreactive (IR) oligodendrocytes were observed throughout the white matter. Furthermore, numerous ectopic substance P (SP)- and calcitonin gene-related peptide (CGRP)-IR fibres were detected in the white matter of the spinal cord of transgenic mice. NGF-IR oligodendrocytes and ectopic SP- and CGRP- fibres were entirely absent from control mice. In the cervical and lumbar dorsal root ganglia, the percentages of SP-IR neurons were significantly higher in transgenic mice when compared with controls. At the electron microscope level, ectopic SP- and CGRP-IR fibres were characterized as unmyelinated axons and axonal boutons. SP co-localized with CGRP in some of those axonal boutons and fibres. Capsaicin treatment of adult mice completely abolished the ectopic SP-IR fibres, confirming their primary sensory origin. Our results indicate that primary sensory neurons are responsive to NGF over-expression in the CNS. Ectopic SP- and CGRP-IR fibres in the white matter are likely to represent collateral sprouts of the central processes of the dorsal root ganglion cells which were triggered by NGF over-expressed in the myelinating oligodendrocytes in the spinal cord of transgenic mice.     

单侧隐睾大鼠生殖股神经神经核改变

目的 研究在单侧隐睾大鼠背根神经节中,生殖股神经神经核的改变.方法 应用辣根过氧化酶(HRP)逆行示踪与免疫荧光相结合的双重技术,观察注射17-β雌二醇获得隐睾大鼠动物模型GFN传导及其递质免疫反应细胞的变化.取单侧隐睾大鼠12只设为A组;取12只单侧隐睾大鼠在出生后第30天切断隐睾侧生殖股神经(GFN)设为B组;另取正常同日龄大鼠10只设为C组.分别在日龄第58天隐睾侧GFN末梢用HRP逆行示踪.48 h后对SD大鼠灌注固定后,取大鼠的T12～L3脊髓及背根神经结,脱水后冰冻切片显色,并用免疫荧光标记CGRP.结果 HRP标记的生殖股神经在L1,L2水平的背根神经节感觉核中发现,A组HRP阳性神经元表达平均灰度和阳性单位较B组和C组要高有统计学意义(P<0.05),B组和C组比较(P＞0.05);CGRP免疫荧光结果中A组较B组、C组表达要少(P<0.05).结论 单侧隐睾大鼠生殖股神经感觉核神经元增多,运动核相对减少,分泌的神经递质CGPR减少.     

Biphasic Response of Spinal GABAergic Neurons after a Lumbar Rhizotomy in the Adult Rat

The expression of γ-aminobutyric acid (GABA) and of the isoforms of the enzyme involved in its synthesis, glutamic acid decarboxylase (GAD), is modified in several rat brain structures in different injury models. The aim of the present work was to determine whether such plasticity of the GABAergic system also occurred in the deafferented adult rat spinal cord, a model where a major reorganization of neural circuits takes place. GABAergic expression following unilateral dorsal rhizotomy was studied by means of non-radioactive in situ hybridization to detect GADs₆₇ mRNA and by immunohistochemistry to detect GAD₆₇ protein and GABA. Three days following rhizotomy the number of GAD₆₇ mRNA-expressing neurons was decreased in the superficial layers of the deafferented horn, while GABA immunostaining of axonal fibres located in this region was highly increased. Seven days after lesion, on the other hand, many GAD₆₇ mRNA-expressing neurons were bilaterally detected in deep dorsal and ventral layers, this expression being correlated with the increased detection of GADs₆₇ immunostained somata and with the reduction of GABA immunostaining of axons. GABA immunostaining was frequently found to be associated with reactive astrocytes that exhibited intense immunostaining for glial fibrillary acidic protein (GFAP) but remained GADs₆₇ negative. These results indicate that degeneration of afferent terminals induces a biphasic response of GABAergic spinal neurons located in the dorsal horn and show that many spinal neurons located in deeper regions re-express GAD₆₇, suggesting a possible participation of the local GABAergic system in the reorganization of disturbed spinal networks.     

Growth of myelinated lumbosacral ventral root fibers in kittens after early postnatal dorsal rhizotomy,dorsal rhizotomy and spinal cord hemisection,or sciatic neurectomy

The fiber-size distribution of myelinated lumbosacral ventral root fibers was studied in 14 kittens at various survival times after unilateral transection of corresponding dorsal roots at 8 days of age. In six animals an ipsilateral spinal cord hemisection was made in addition to dorsal rhizotomy. For comparison, two kittens were subjected instead to a unilateral sciatic neurectomy. The size spectra obtained from the ventral roots on the side of the lesion were compared with the fiber-size distribution in the same roots on the side not affected by the operation. In addition, three normal kittens were used as control animals. The findings show that neither the dorsal rhizotomy nor the combined dorsal rhizotomy and spinal cord hemisection had any measurable effect on the postnatal growth of the myelinated fibers in the relevant roots. The lesions resulted in a grave motor deficit which suggests that a normal function is not of any major importance for the growth in fiber size. After sciatic neurectomy the size spectrum of the myelinated fibers in ventral roots L7 and S1 showed a distinct shift to the left indicating a more severe retrograde degeneration of large than of small ventral horn neurons.     

Crossed and uncrossed projections to the cat sacrocaudal spinal cord: III. Axons expressing calcitonin gene-related peptide immunoreactivity

We have investigated the projection patterns of peptidergic small-diameter primary afferent fibers to the cat sacrocaudal spinal cord, a region associated with midline structures of the lower urogenital system and of the tail. Calcitonin gene-related peptide (CGRP)-immunoreactive (CGRP-IR) primary afferent fibers were observed within the superficial laminae, rostrally as the typical inverted U-shaped band that capped the separate dorsal horns (S1 to rostral S2) and caudally as a broad band that spanned the entire mediolateral extent of the fused dorsal horns (caudal S2 and caudal). Within the dorsal gray commissure, labeling was seen as a periodic vertical, midline band. CGRP-IR labeling was prevalent in an extensive mediolateral distribution at the base of the dorsal horn, originating from both lateral and medial collateral bundles that extend from the superficial dorsal horn. Some bundles, in part traveling within the dorsal commissure, conspicuously crossed the midline. In addition to the robust projection to the superficial dorsal horn, there was a more extensive distribution of CGRP-IR fibers within the deeper portions of the cat sacrocaudal dorsal horn than has been reported for other regions of the cat spinal cord. Presumably, these deep projections convey visceral information to projection or segmental neurons at the neck of the dorsal horn and in the region of the central canal. This deep distribution overlaps the reported projections of the pelvic and pudendal nerves. In addition, the contralateral projections of CGRP-IR fibers may form an anatomical substrate of the bilateral receptive fields for selective dorsal horn neurons. The density and variety of CGRP-IR projection patterns is a reflection of the functional attributes of the innervated structures.     

Effect of Brn-3a deficiency on CGRP-immunoreactivity in the dorsal root ganglion

Immunohistochemistry for calcitonin gene-related peptide (CGRP) was performed on the dorsal root ganglion (DRG) and spinal cord in wildtype and knockout mice for Brn-3a. CGRP-immunoreactive (-IR) neurons were abundant in the DRG of wildtype, heterozygous and knockout mice. Cell size analysis revealed that CGRP-IR neurons were of various sizes in wildtype and heterozygous mice. In the knockout mice, however, most of CGRP-IR neurons were small. In the spinal cord of knockout mice, the number of CGRP-IR fibers increased in the dorsal column but decreased in the deep part of the dorsal horn. The loss of Brn-3a may have different effects on CGRP-IR expression in small and large DRG neurons.     

Phosphorylated MAP1B is induced in central sprouting of primary afferents in response to peripheral injury but not in response to rhizotomy

A peripheral nerve lesion induces sprouting of primary afferents from dorsal root ganglion (DRG) neurons into lamina II of the dorsal horn. Modifications of the environment in consequence to the axotomy provide an extrinsic stimulus. A potential neuron-intrinsic factor that may permit axonal sprouting is microtubule-associated protein 1B (MAP1B) in a specific phosphorylated form (MAP1B-P), restricted to growing or regenerating axons. We show here that both in rat and mouse, a sciatic nerve cut is rapidly followed by the appearance of MAP1B-P expression in lamina II, increasing to a maximum between 8 and 15 days, and diminishing after three months. Evidence is provided that sprouting and induction of MAP1B-P expression after peripheral injury are phenomena concerning essentially myelinated axons. This is in accordance with in situ hybridization data showing especially high MAP1B-mRNA levels in large size DRG neurons that give rise to myelinated fibers. We then employed a second lesion model, multiple rhizotomy with one spared root. In this case, unmyelinated CGRP expressing fibers do indeed sprout, but coexpression of MAP1B-P and CGRP is never observed in lamina II. Finally, because a characteristic of myelinated fibers is their high content in neurofilament protein heavy subunit (NF-H), we used NF-H-LacZ transgenic mice to verify that MAP1B-P induction and central sprouting were not affected by perturbing the axonal organization of neurofilaments. We conclude that MAP1B-P is well suited as a rapidly expressed, axon-intrinsic marker associated with plasticity of myelinated fibers.