Semaphorin and neuropilin expression in motoneurons after intraspinal motoneuron axotomy.

We have examined mRNA and protein distribution for the axon guidance molecules semaphorin3A, 3F, 4F and semaphorin receptors neuropilin-1 and 2, 1-21 days after intramedullary axotomy of rat lumbar spinal cord motoneurons. We show that semaphorin3A mRNA and protein are up-regulated in the scar and in motoneurons from 3 days and upto 3 weeks after injury. Neuropilin-1 mRNA showed no changed expression in axotomized motoneurons. Semaphorin3F mRNA expression was found in ventral roots after ventral funiculus lesion (VFL) and neuropilin-2 mRNA was found in affected motoneurons from 1 day after injury throughout the examined period. Semaphorin4F mRNA was first found in motoneurons 3 weeks after lesion. These results suggest semaphorin/neuropilin involvement in the injury response of intramedullary axotomized motoneurons.

Expression of neuregulin and ErbB3 and ErbB4 after a traumatic lesion in the ventral funiculus of the spinal cord and in the intact primary olfactory system.

Neuron-derived neuregulins have been implicated in the regulation of glial cell function and survival. This factor family and its receptors may therefore be assumed to be of importance for the cellular response to traumatic injury. In this study we have examined the distribution of mRNA for neuregulin 1 (NRG1), ErbB3 and ErbB4-receptor tyrosine kinases after a ventral funiculus lesion in the lumbar spinal cord (VFL). The techniques used were in situ hybridization and immunohistochemistry. The survival times were 1-21 days. The spinal cords from normal adult and embryonic rats were used as controls. For comparison, sections from the olfactory bulb of perinatal and adult rats were also included in the study. Expression of NRG1 mRNA was observed in motoneurons in the intact spinal cord. A decrease in the labeling for NRG1 mRNA was seen during the first 5 days after VFL but then became slightly upregulated at 3 weeks after the lesion. A high labeling signal for ErbB3-mRNA was observed in the ventral and dorsal roots of E16 and E18 embryos. Labeling for ErbB3-mRNA was strong in the affected ventral root at 3 days after the VFL, reached a maximum at 1 week and was still upregulated after 3 weeks. Increased labeling for ErbB3 was also noted in scattered cells in the scar tissue 1-3 weeks after the VFL. These findings were verified with immunohistochemistry for ErbB3. A strong labeling for ErbB3 in the olfactory nerve fiber layer and olfactory nerve bundles was observed in rats of all ages examined. ErbB4 had strong expression in the embryonic spinal cord, but no evidence for lesion-induced regulation of ErbB4 receptors could be found after the VFL. Our data show that ErbB3 in the ventral roots was upregulated after a VFL and that NRG1 mRNA was initially downregulated in the motoneurons. The lesion-induced changes in the expression of NRG1 and ErbB3 in the injured spinal cord and denervated ventral root can be assumed to be of importance for axonal growth and the regulation of glial cell survival.

Properties of motoneurons underlying their regenerative capacity after axon lesions in the ventral funiculus or at the surface of the spinal cord.

Spinal motoneurons represent neurons with axons located in both the central (CNS) and peripheral (PNS) nervous systems. Following a lesion to their axons in the PNS, motoneurons are able to regenerate. The regenerative capacity of these neurons is seen also after lesion in the ventral funiculus of the spinal cord, i.e. within the CNS compartment. Thus, after an axotomy within the ventral funiculus, motoneurons respond with a changing polarity towards production of axons, sometimes even from the dendritic tree. This capacity can be used in cases of ventral root avulsion (VRA) lesions, if a conduit for outgrowing axons is presented in the form of replanted ventral roots. In human cases, this procedure may accomplish return of function in denervated muscles. The strong regenerative capacity of motoneurons provides the basis for studies of the response in motoneurons with regard to their contents of substances related to survival and regeneration. Such studies have shown that, of the large number of receptors for neurotrophic substances and extracellular matrix molecules, mRNAs for receptors or receptor components for neurotrophin-3 (NT-3), ciliary neurotrophic factor (CNTF) and leukemia inhibitory factor (LIF) are strongly downregulated after VRA, while receptors for glial cell line-derived neurotrophic factor (GDNF) and laminins are profoundly upregulated. These results should be considered in the design of combined pharmacological and surgical approaches to lesions of motor axons at or close to the CNS-PNS interface.

Expression of tenascin R and J1 mRNA in motoneurons after a traumatic lesion in the spinal cord.

We demonstrate, using in situ hybridization, that mRNA for the anti-adhesive molecules tenascin R and J1 in the adult rat spinal motoneurons are down-regulated rapidly as a reaction after a ventral funiculus lesion. Tenascin-R was significantly down-regulated at day 1 and normalized after 3 weeks. Tenascin-J1 declined to its lowest value at day 3 and returned to the initial level after 3 weeks. In adjacent sections, the distribution of macrophages was studied with immuno histochemistry. The density of macrophages reached a maximum 3 days after the injury. Thus, the density of macrophages appeared to be inversely related to the level of tenascin mRNA. These data are compatible with the notion that neuronal tenascins may modulate the adhesion of perincurial inflammatory cells.

Regulation of laminin-associated integrin subunit mRNAs in rat spinal motoneurons during postnatal development and after axonal injury.

Two important prerequisites for successful axon regeneration are that appropriate extracellular molecules are available for outgrowing axons and that receptors for such molecules are found in the regenerating neuron. Laminins and their receptors in the integrin family are examples of such molecules, and laminin-associated integrin subunits alpha 3, alpha 6, alpha 7, and beta 1 mRNAs have all been detected in adult rat motoneurons. We have here, by use of in situ hybridization histochemistry, examined the normal postnatal development of the expression in motoneurons of these mRNAs and integrin beta 4 mRNA, all of which have been associated with laminin-2. We studied the regulation of these mRNAs, 1-42 days after two types of axotomy in the adult rat (sciatic nerve transection, SNT; ventral root avulsion, VRA) and 1-10 days after SNT in the neonatal animal. During postnatal development, there was a distinct shift in the integrin composition from a stronger expression of the alpha 6 subunit to a very clear dominance of alpha 7 in the adult. All types of axotomy in the adult rat induced initial (1-7 days) large up-regulations of alpha 6, alpha 7 and beta1 subunit mRNAs (250-500%). Only minor changes for alpha 3 mRNA were seen, and beta 4 mRNA could not be detected at all in motoneurons. After adult SNT, the alpha 7 and beta 1 subunits were up-regulated throughout the studied period, and the alpha 6 subunit mRNA was eventually normalized. After VRA, however, the alpha 7 and beta1 levels peaked earlier than after SNT and were normalized at 42 days, whereas alpha 6 mRNA was up-regulated longer than after SNT. Neonatal SNT had much smaller effects on the expression of the studied subunits. The results suggest that an important part of the response to axotomy of motoneurons is to up-regulate receptors for laminin. The developmental shift in integrin subunit composition and the various responses seen in the lesion models indicate that different isoforms of laminin play a role in the regenerative response.

Differential expression of tenascin-C, tenascin-R, tenascin/J1, and tenascin-X in spinal cord scar tissue and in the olfactory system.

The members of the tenascin family are involved in a number of developmental processes, mainly by their ability to regulate cell adhesion. We have here studied the distribution of mRNAs for tenascin-X, -C, and -R and the closely related molecule tenascin/J1 in the olfactory system and spinal cord. The olfactory bulb and nasal mucosa were studied during late embryonic and early postnatal development as well as in the adult. The spinal cord was studied during late embryonic development and after mechanical lesions. In the normal rat, the spinal cord and olfactory bulb displayed similar patterns of tenascin expression. Tenascin-C, tenascin-R, and tenascin/J1 were all expressed in the olfactory bulb and spinal cord during development, while tenascin/J1 was the only extensively expressed tenascin molecule in the adult. In both regions tenascin/J1 was expressed in both nonneuronal and neuronal cells. After a spinal cord lesion, mRNAs for tenascin-C, -X, -R, and/J1 were all upregulated and had their own specific spatial and temporal expression patterns. Thus, even if axonal outgrowth occurs to some extent both in the adult rat primary olfactory system and in spinal cord scar tissue after lesion, the tenascin expression patterns in these two situations are totally different.

Differential regulation of trophic factor receptor mRNAs in spinal motoneurons after sciatic nerve transection and ventral root avulsion in the rat.

After sciatic nerve lesion in the adult rat, motoneurons survive and regenerate, whereas the same lesion in the neonatal animal or an avulsion of ventral roots from the spinal cord in adults induces extensive cell death among lesioned motoneurons with limited or no axon regeneration. A number of substances with neurotrophic effects have been shown to increase survival of motoneurons in vivo and in vitro. Here we have used semiquantitative in situ hybridization histochemistry to detect the regulation in motoneurons of mRNAs for receptors to ciliary neurotrophic factor (CNTF), leukemia inhibitory factor (LIF), glial cell line-derived neurotrophic factor (GDNF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3) 1-42 days after the described three types of axon injury. After all types of injury, the mRNAs for GDNF receptors (GFRalpha-1 and c-RET) and the LIF receptor LIFR were distinctly (up to 300%) up-regulated in motoneurons. The CNTF receptor CNTFRalpha mRNA displayed only small changes, whereas the mRNA for membrane glycoprotein 130 (gp130), which is a critical receptor component for LIF and CNTF transduction, was profoundly down-regulated in motoneurons after ventral root avulsion. The BDNF full-length receptor trkB mRNA was up-regulated acutely after adult sciatic nerve lesion, whereas after ventral root avulsion trkB was down-regulated. The NT-3 receptor trkC mRNA was strongly down-regulated after ventral root avulsion. The results demonstrate that removal of peripheral nerve tissue from proximally lesioned motor axons induces profound down-regulations of mRNAs for critical components of receptors for CNTF, LIF, and NT-3 in affected motoneurons, but GDNF receptor mRNAs are up-regulated in the same situation. These results should be considered in relation to the extensive cell death among motoneurons after ventral root avulsion and should also be important for the design of therapeutical approaches in cases of motoneuron death.

Induction of VEGF and VEGF receptors in the spinal cord after mechanical spinal injury and prostaglandin administration.

Vascular endothelial growth factor (VEGF) is an angiogenetic factor that promotes endothelial cell proliferation during development and after injury to various types of tissue, including the central nervous system (CNS). Using immunohistochemical and in situ hybridization methods we have here demonstrated that VEGF and its receptors Flk-1, Flt-1 and Neuropilin-1 mRNAs and proteins are induced after incisions in the rat spinal cord. The inducible enzyme for prostaglandin synthesis cyclooxygenase-2 (COX-2) is known to be upregulated after spinal injury, cerebral ischemia and to stimulate angiogenesis. To test the hypothesis that prostaglandins may be involved in the VEGF response after lesion we investigated whether intraspinal microinjections of prostaglandin F2alpha (PGF2alpha) alters VEGF expression in the spinal cord. Such treatment was followed by a strong upregulation of VEGF mRNA and protein in the injection area. Finally, by use of an in vitro model with cell cultures of meningeal fibroblast and astrocyte origin, resembling the lesion area cellular content after spinal cord injury but devoid of inflammatory cells, we showed that VEGF is expressed in this in vitro model cell system after treatment with PGF2alpha and prostaglandin E2 (PGE2). These data suggest that cells within a lesion area in the spinal cord are capable of expressing VEGF and its receptors in response to mechanical injury and that prostaglandins may induce VEGF expression in such cells, even in the absence of inflammatory cells.

Ultrastructural evidence for a preferential elimination of glutamate-immunoreactive synaptic terminals from spinal motoneurons after intramedullary axotomy.

After axotomy in the ventral funiculus of the cat spinal cord, about half of the population of lesioned motoneurons die at 1-3 weeks postoperatively, whereas the other half survives and generates new axons through the lesion area. To identify conditions that may promote survival and regeneration of motoneurons subjected to this kind of injury, the authors examined ultrastructurally lesion-induced changes in the number and distribution of nerve terminals on the somata and proximal dendrites of alpha-motoneurons in the 7th lumbar spinal segment (L7) of the cat spinal cord. Intramedullary axotomy resulted in a profound reduction in the number of nerve terminals impinging on the somata and proximal dendrites, with the maximal effect seen at 3 weeks postlesion. At that time, only 12-25% of the normal number of terminals remained on the cell somata, and 22-33% remained on proximal dendrites. Thereafter, a gradual increase in terminal numbers occurred, reaching normal levels at 34 weeks after the lesion. Already at 2 days postoperatively and, most obviously, at 3 weeks postoperatively, type S nerve terminals were eliminated to a larger degree than type F terminals. Postembedding immunohistochemistry confirmed that the largest reduction at 3 weeks was seen for excitatory glutamate-immunopositive type S nerve terminals (90%), whereas inhibitory glycine-immunoreactive and gamma-aminobutyric acid (GABA)-immunoreactive type F terminals were affected less (70% reduction). This led to a distinct shift in the ratio between the numbers of terminals that were immunopositive for glycine and GABA and the numbers of terminals that were labeled for glutamate. For the cell body, this ratio increased from 3.7 in normal material to 14.5 in lesioned motoneurons, whereas the corresponding values for proximal dendrites were 3.8 and 7.5. The preferential elimination of glutamatergic inputs to lesioned motoneurons may reflect an active reorganization of the synaptic input to diminish the excitotoxic influence on these neurons, thereby promoting the survival of motoneurons after intramedullary axotomy.

Neuroprotection by encephalomyelitis: rescue of mechanically injured neurons and neurotrophin production by CNS-infiltrating T and natural killer cells.

In experimental autoimmune encephalomyelitis (EAE), CD4(+) self-reactive T cells target myelin components of the CNS. However, the consequences of an autoaggressive T cell response against myelin for neurons are currently unknown. We herein demonstrate that EAE induced by active immunization with an encephalitogenic myelin basic protein peptide dramatically reduces the loss of spinal motoneurons after ventral root avulsion in rats. Both brain-derived neurotophic factor (BDNF)- and neurotrophin-3 (NT-3)-like immunoreactivities were detected in mainly T and natural killer (NK) cells in the spinal cord. In addition, very high levels of BDNF, NT-3, and glial cell line-derived neurotrophic factor mRNAs were present in T and NK cell populations infiltrating the CNS. Interestingly, bystander recruited NK and T cells displayed similar or higher neurotrophic factor levels compared with the EAE disease-driving encephalitogenic T cell population. High levels of tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) mRNAs were also detected, and both these cytokines can be harmful to several types of CNS cells, including neurons. However, treatment of embryonic motoneuron cultures with TNF-alpha or IFN-gamma only had a deleterious effect in cultures deprived of neurotrophic factors. These results suggest that the potentially neurodamaging consequences of severe CNS inflammation are curbed by the production of several potent neurotrophic factors in leukocytes.

Developmental and lesion-induced changes in the distribution of the glucose transporter Glut-1 in the central and peripheral nervous system.

The active transport of D-glucose from the vascular to the neural compartment requires the presence of a carrier molecule at the blood-brain and the blood-nerve barrier. The glucose transporter 1 (Glut-1) seems to be the main carrier in blood-tissue barriers of endothelial and perineurial type. The distribution of Glut-1 was assessed in the normal central and peripheral nervous system of young and adult animals and compared with changes after nerve injury. Immuno-histochemistry, in situ hybridization, and perfusions with Evans Blue were carried out. Glut- I was not expressed in the perineurium of peripheral nerves at birth, but appeared in the perineurium of peripheral nerves, spinal roots, in the capsule of dorsal-root ganglia, and in the pia mater of adult animals. The perineurium of peripheral nerves subjected to Wallerian degeneration presented a faint Glut-1 immunoreaction, which was restored after regeneration. Glut-1 was expressed in capillaries of the gray substance of the spinal cord. Perineurial-derived lamellar cells of Pacinian corpuscles exhibited a strong Glut-1-like immunoreactivity in response to denervation and during development. Merkel cells and Meissner corpuscles were found to be Glut-1 negative. Glut-1 seems to reflect the quality of an adult, mature perineurial and blood-nerve barrier.

Multiple messengers in descending serotonin neurons: localization and functional implications.

In the present review article we summarize mainly histochemical work dealing with descending bulbospinal serotonin neurons which also express a number of neuropeptides, in particular substance P and thyrotropin releasing hormone. Such neurons have been observed both in rat, cat and monkey, and may preferentially innervate the ventral horns of the spinal cord, whereas the serotonin projections to the dorsal horn seem to lack these coexisting peptides. More recent studies indicate that a small population of medullary raphe serotonin neurons, especially at rostral levels, also synthesize the inhibitory neurotransmitter gamma-amino butyric acid (GABA). Many serotonin neurons contain the glutamate synthesizing enzyme glutaminase and can be labelled with antibodies raised against glutamate, suggesting that one and the same neuron may release several signalling substances, causing a wide spectrum of post- (and pre-) synaptic actions.

Axon regeneration of spinal motoneurons following a lesion at the cord-ventral root interface.

Those insults to the spinal cord which occur when ventral or dorsal roots are avulsed from the surface of the cord have been considered unfavourable with regard to both survival and axon regeneration of lesioned neurons. In this review, we describe the development of a surgical procedure aiming at a restoration of motor function after ventral root avulsion lesions. This development includes a series of investigations in animals, where an unexpected capacity for cell survival and axon regeneration of motoneurons after a cut lesion in the spinal cord was demonstrated and analyzed in great detail. Based on these findings, a surgical technique was tested, where avulsed ventral roots were replanted into the cord. After confirmation that such implanted roots could serve as a conduit for outgrowing motor axons in animals, the technique has been evaluated in a limited number of human cases of root avulsion lesions. We conclude that surgical intervention may indeed lead to return of motor function also in human cases of ventral root avulsion lesions. Interestingly, the procedure also seems to have an attenuating effect on the pain that develops in cases with a combined dorsal root avulsion. Lastly, we conclude that the cut lesion in the ventral part of the spinal cord, followed by axon regeneration in motoneurons may serve as a model for axon regeneration in the central nervous system.

Differential expression of nerve terminal protein isoforms in VAChT-containing varicosities of the spinal cord ventral horn.

Of the different types of synaptic contacts with the mammalian spinal motoneuron, the synapse made by the cholinergic, so-called C-terminal of unknown origin and function has special morphological characteristics. Thus, in this synapse, there is no postsynaptic density but rather a large subsynaptic cistern in the motoneuron. To see whether this particular arrangement imposes special demands on the transmitter release machinery, we examined the presence of nerve terminal proteins in the C-terminal by using immunohistochemistry. Cholinergic nerve fibers and terminals in the spinal cord ventral horn were identified with an antiserum to the vesicular acetylcholine transporter (VAChT) protein. Immunohistochemistry in combination with confocal laser microscopy showed the presence of synaptosomal-associated protein of 25 kDa (SNAP-25)-, syntaxin-, cysteine string protein (CSP)-, synuclein-, synapsin I-, synapsin I/II-, synaptotagmin I-, synaptotagmin I/II-, synaptophysin-, and synaptobrevin-2-like immunoreactivity (-LI) in VAChT-containing C-terminals. Synaptotagmin III and synaptobrevin 1 could not be demonstrated in this type of terminal. VAChT-containing varicosities in the Renshaw cell area, with a probable origin from motoneuron axon collaterals, exhibited CSP, synapsin I/II, and synaptobrevin-1-LI, but not SNAP-25-, syntaxin-, synuclein-, synapsin I-, synaptotagmin I-, synaptotagmin I/II-, synaptophysin- and synaptobrevin-2-LI. The results suggest a differential content of nerve terminal proteins and their isoforms in cholinergic C-terminals apposing motoneurons and in the Renshaw cell area. It is concluded that C-terminals contain synaptic proteins necessary for fast transmitter release, and their origin should not be the motoneurons themselves.

Expression of insulin-like growth factors and corresponding binding proteins (IGFBP 1-6) in rat spinal cord and peripheral nerve after axonal injuries.

Insulin-like growth factors (IGFs) exert trophic effects on several different cell types in the nervous system, including spinal motoneurons. After peripheral nerve injury, the increased expression of IGFs in the damaged nerve has been suggested to facilitate axonal regeneration. Here we have examined the expression pattern of mRNAs encoding IGF-1 and and -2, IGF binding proteins (IGFBPs) 1-6 in the rat spinal cord and peripheral nerve in three lesion models affecting lumbar motoneurons, i.e., sciatic nerve transection, ventral root avulsion, and a cut lesion in the ventral funiculus of the spinal cord. The expression was also studied in enriched Schwann cell and astrocyte cultures. The injured sciatic nerve expressed IGF-1 and IGF-2 as well as IGFBP-4 and IGFBP-5, whereas central nervous system (CNS) scar tissue expressed IGF-1, IGFBP-2, and IGFBP-5. IGFBP-6 mRNA was strongly upregulated in spinal motoneurons after all three types of lesions. IGFBP-6-like immunoreactivity was present in motoneuron cell bodies, dendrites in the ventral horn, and axons in the sciatic nerve. In line with the in vivo findings, cultured Schwann cells expressed IGF-1, IGF-2, IGFBP-4, and IGFBP-5 mRNAs, whereas cultured astrocytes expressed IGF-1, IGFBP-2, and IGFBP-5 mRNAs. These findings show that IGF-1 is available for lesioned motoneurons both after peripheral and central axonal lesions, whereas there are clear differences in the expression patterns for IGF-2 and some of the binding proteins in CNS and peripheral nervous system (PNS) scar tissue. The robust upregulation of IGFBP-6 mRNA in lesioned motoneurons suggests that this binding protein may be of special relevance for the severed cells.

Nerve growth factor induces process formation in meningeal cells: implications for scar formation in the injured CNS.

Nerve growth factor (NGF) induces the differentiation and supports the survival of subpopulations of neurons in the PNS and CNS. Here we report that meningeal cells in the pia mater express immunoreactivity and mRNA for both known NGF receptors, the low-affinity receptor p75 and the tyrosine kinase receptor trkA. NGF induces rapid tyrosine phosphorylation of trkA in meningeal cells in vitro. NGF does not stimulate proliferation of primary meningeal cells but induces process outgrowth. p75- and trkA-immunoreactive meningeal cells with long processes, resembling NGF-treated cells in vitro, are abundant in the scar tissue that forms at spinal cord lesions in rat and cat. These data suggest that NGF, which is expressed at increased levels in the brain and spinal cord after lesions, may be involved in scar formation in the injured CNS.

Expression of MHC class I and beta2-microglobulin in rat spinal motoneurons: regulatory influences by IFN-gamma and axotomy.

The low expression of MHC antigens is believed to be one factor of importance contributing to the immune-privileged status of CNS neurons. We here describe that motoneurons, in contrast to other nerve cells in the lumbar spinal cord of the adult rat, express both MHC class I and beta2-microglobulin mRNA. The motoneurons also display in situ hybridization signal for IFN-gamma receptor mRNA. After a peripheral axotomy, the motoneurons show a clear upregulation of beta2-microglobulin mRNA. IFN-gamma treatment of cultured rat embryonic spinal motoneurons causes a similar upregulation of especially beta2-microglobulin. Based on these facts, we propose that spinal motoneurons can be influenced by IFN-gamma and recognized by cytotoxic CD8+ T-cells. These findings could be of relevance in the search for pathogenetic mechanisms in motoneuron-specific diseases, such as ALS.

Changes in the mRNA expression pattern, with special reference to calcitonin gene-related peptide, after axonal injuries in rat motoneurons depends on age and type of injury.

The axotomy reaction in motoneurons after a peripheral nerve transection in the adult animal is characterized by a robust upregulation of alpha-calcitonin gene-related peptide (CGRP) messenger RNA (mRNA) together with mRNAs encoding cytoskeletal and growth-related proteins. Here we have examined whether the nature of the lesion and the age of the animal have any impact on the mRNA regulation in severed cells. Thus, the effect of a sciatic nerve transection in the adult rat was compared with, on the one hand, ventral root avulsions in the adult animal and, on the other hand, sciatic nerve transection in the immature animal. In the two latter cases, a proportion of the lesioned cells die and overall chances of regeneration are small. In the adult animal a sciatic nerve transection induced an upregulation of alpha-CGRP mRNA from the 3rd day after surgery and throughout the first 3 weeks (the time span of the study). Also low-affinity nerve growth factor receptor (p75) and growth-associated protein-43 (GAP-43) mRNAs were upregulated during the entire 3-week period. In contrast, after ventral root avulsion, the expression of alpha-CGRP, c-jun, and p75 mRNAs were normalized within the 1st postoperative week, while GAP-43 mRNA was still upregulated at 3 weeks. Galanin message-associated peptide (GMAP) mRNA became upregulated preferentially in motoneurons subjected to ventral root avulsion, while nitric oxide synthase (NOS) mRNA was expressed exclusively after the latter type of injury. In the immature animal, alpha-CGRP mRNA was downregulated after sciatic nerve transection in rats aged 3 days or 7 days at the time of surgery; while, in contrast, an upregulation was seen in 12- or 21-day-old animals. GAP-43 and c-jun mRNAs were upregulated in lesioned motoneurons of all ages, while GMAP mRNA was upregulated preferentially in lesioned motoneurons of early postnatal animals. p75 mRNA was expressed in unlesioned immature motoneurons until the age of 7-10 days. The downregulation of p75 mRNA in intact cells at this age coincided with a developmental switch in the ability of axotomized cells to express increased levels of p75 mRNA. No expression of NOS mRNA was detectable in lesioned cells of any of the age groups. These results show that the age of the animal and the type of axonal injury are indeed to a high degree influencing the changes seen in the protein expression pattern in axotomized rat motoneurons. The different responses in these paradigms suggest differences in the trophic response from surrounding glia or the trophic responsiveness of lesioned motoneurons. Also, the results may indicate different roles for the studied substances during the regenerative response of lesioned neurons. Of the substances studied here, upregulation of alpha-CGRP and p75 mRNAs best correlated with a possibility of axon regeneration.

Distribution of glutamate-, glycine- and GABA-immunoreactive nerve terminals on dendrites in the cat spinal motor nucleus.

The dendritic tree constitutes more than 93% of the receptive membrane area of a spinal motoneuron, yet little is known about its synaptic inputs. In this study we examined the distribution of glutamate-, GABA- and glycine-like immunoreactivity in boutons apposing dendrites in the L7 spinal cord motor nucleus, by use of postembedding immunohistochemistry on serial sections. We examined 799 boutons apposing 401 cross-sectioned dendrites of different calibre (range 0.2-15 microm), and 14 first-order (stem) dendrites. Thirty-five percent (35%) of the boutons were immunopositive for glutamate and 59% for GABA and/or glycine. Among the latter, 30% showed glycine immunoreactivity only and 24% were immunoreactive for both GABA and glycine. Very few were immunoreactive only for GABA (5%). As few as 6% of the boutons were judged as not enriched for any amino acid analysed. The fine structural characteristics of the boutons were in accordance with previous descriptions. The sample of dendrites was arranged in calibre bins in order to facilitate distribution analysis. Stem dendrites differed from the other bins, with a high total bouton covering (61%) and a high bouton density. Sixty-nine percent of the membrane covering was by glycine- and/or GABA-immunoreactive boutons, whereas 18% was covered by boutons enriched in glutamate. For non-stem dendrites, bouton covering fell from 33% to 12% with decreasing calibre. However, bouton apposition length decreased in parallel, yielding a fairly uniform bouton density among dendrites of different calibre. The lack of correlation between packing density and dendrite calibre was also evident when the sample of dendrites was broken down into subsamples based on content of amino acid immunoreactivity. The latter analysis also revealed that both the relative covering and density of boutons containing inhibitory amino acids (57%; glycine and/or GABA) and glutamate (38%), respectively, did not vary systematically with dendrite calibre. Combined, the data indicate that in non-stem dendrites the proportion of excitatory and inhibition inputs does not change systematically throughout the dendritic arborizations of spinal alpha-motoneurons. Thus, spinal motoneurons can, with respect to the general synaptic architecture, be divided into two main compartments, i.e. the proximal soma-juxtasomatic compartment (including stem dendrites) and the distal dendritic compartment. The proximal domain is under a powerful glycine and/or GABA influence. Finally, based on the data presented here and previously published data, it was calculated that spinal alpha-motoneurons receive in the range of 50-140 x 10(3) synaptic boutons.