Unbiased expression mapping identifies a link between the complement and cholinergic systems in the rat central nervous system.

The complement system is activated in a wide spectrum of CNS diseases and is suggested to play a role in degenerative phenomena such as elimination of synaptic terminals. Still, little is known of mechanisms regulating complement activation in the CNS. Loss of synaptic terminals in the spinal cord after an experimental nerve injury is increased in the inbred DA strain compared with the PVG strain and is associated with expression of the upstream complement components C1q and C3, in the absence of membrane attack complex activation and neutrophil infiltration. To further dissect pathways regulating complement expression, we performed genome-wide expression profiling and linkage analysis in a large F2(DA × PVG) intercross, which identified quantitative trait loci regulating expression of C1qa, C1qb, C3, and C9. Unlike C1qa, C1qb, and C9, which all displayed distinct coregulation with different cis-regulated C-type lectins, C3 was regulated in a coexpression network immediately downstream of butyrylcholinesterase. Butyrylcholinesterase hydrolyses acetylcholine, which exerts immunoregulatory effects partly through TNF-α pathways. Accordingly, increased C3, but not C1q, expression was demonstrated in rat and mouse glia following TNF-α stimulation, which was abrogated in a dose-dependent manner by acetylcholine. These findings demonstrate new pathways regulating CNS complement expression using unbiased mapping in an experimental in vivo system. A direct link between cholinergic activity and complement activation is supported by in vitro experiments. The identification of distinct pathways subjected to regulation by naturally occurring genetic variability is of relevance for the understanding of disease mechanisms in neurologic conditions characterized by neuronal injury and complement activation.

Complement receptor 2 is up regulated in the spinal cord following nerve root injury and modulates the spinal cord response.

BACKGROUND: Activation of the complement system has been implicated in both acute and chronic states of neurodegeneration. However, a detailed understanding of this complex network of interacting components is still lacking. METHODS: Large-scale global expression profiling in a rat F2(DAxPVG) intercross identified a strong cis-regulatory influence on the local expression of complement receptor 2 (Cr2) in the spinal cord after ventral root avulsion (VRA). Expression of Cr2 in the spinal cord was studied in a separate cohort of DA and PVG rats at different time-points after VRA, and also following sciatic nerve transection (SNT) in the same strains. Consequently, Cr2 (-/-) mice and Wt controls were used to further explore the role of Cr2 in the spinal cord following SNT. The in vivo experiments were complemented by astrocyte and microglia cell cultures. RESULTS: Expression of Cr2 in naïve spinal cord was low but strongly up regulated at 5-7 days after both VRA and SNT. Levels of Cr2 expression, as well as astrocyte activation, was higher in PVG rats than DA rats following both VRA and SNT. Subsequent in vitro studies proposed astrocytes as the main source of Cr2 expression. A functional role for Cr2 is suggested by the finding that transgenic mice lacking Cr2 displayed increased loss of synaptic nerve terminals following nerve injury. We also detected increased levels of soluble CR2 (sCR2) in the cerebrospinal fluid of rats following VRA. CONCLUSIONS: These results demonstrate that local expression of Cr2 in the central nervous system is part of the axotomy reaction and is suggested to modulate subsequent complement mediated effects.

Mitofusin 2 is necessary for striatal axonal projections of midbrain dopamine neurons.

Mitochondrial dysfunction is implicated in aging and degenerative disorders such as Parkinson's disease (PD). Continuous fission and fusion of mitochondria shapes their morphology and is essential to maintain oxidative phosphorylation. Loss-of-function mutations in PTEN-induced kinase1 (PINK1) or Parkin cause a recessive form of PD and have been linked to altered regulation of mitochondrial dynamics. More specifically, the E3 ubiquitin ligase Parkin has been shown to directly regulate the levels of mitofusin 1 (Mfn1) and Mfn2, two homologous outer membrane large GTPases that govern mitochondrial fusion, but it is not known whether this is of relevance for disease pathophysiology. Here, we address the importance of Mfn1 and Mfn2 in midbrain dopamine (DA) neurons in vivo by characterizing mice with DA neuron-specific knockout of Mfn1 or Mfn2. We find that Mfn1 is dispensable for DA neuron survival and motor function. In contrast, Mfn2 DA neuron-specific knockouts develop a fatal phenotype with reduced weight, locomotor disturbances and death by 7 weeks of age. Mfn2 knockout DA neurons have spherical and enlarged mitochondria with abnormal cristae and impaired respiratory chain function. Parkin does not translocate to these defective mitochondria. Surprisingly, Mfn2 DA neuron-specific knockout mice have normal numbers of midbrain DA neurons, whereas there is a severe loss of DA nerve terminals in the striatum, accompanied by depletion of striatal DA levels. These results show that Mfn2, but not Mfn1, is required for axonal projections of DA neurons in vivo.

Neuronal myosin-X is upregulated after peripheral nerve injury and mediates laminin-induced growth of neurites.

The successful outcome of peripheral neuronal regeneration is attributed both to the growth permissive milieu and the intrinsic ability of the neuron to initiate appropriate cellular responses such as changes in gene expression and cytoskeletal rearrangements. Even though numerous studies have shown the importance of interactions between the neuron and the extracellular matrix (ECM) in axonal outgrowth, the molecular mechanisms underlying the contact between ECM receptors and the cellular cytoskeleton remain largely unknown. Unconventional myosins constitute an important group of cytoskeletal-associated motor proteins. One member of this family is the recently described myosin-X. This protein interacts with several members of the axon growth-associated ECM receptor family of integrins and could therefore be important in neuronal outgrowth. In this study, using radioactive in situ hybridization, we found that expression of myosin-X mRNA is upregulated in adult rat sensory neurons and spinal motoneurons after peripheral nerve injury, but not after central injury. Thus, myosin-X was upregulated after injuries that can be followed by axonal regeneration. We also found that the protein is localized to neuronal growth cones and that silencing of myosin-X using RNA interference impairs the integrin-mediated growth of neurites on laminin, but has no effect on non-integrin mediated growth on N-cadherin.

Evidence of hypothalamic degeneration in the anorectic anx/anx mouse.

Mice homozygous for the anorexia (anx) mutation are characterized by poor food intake and death by three to five weeks after birth. By P21 these mice display lower density of hypothalamic neuropeptides, including Agouti gene-related protein (AGRP). The AGRP/neuropeptide Y (NPY) system of the anx/anx mice develops normally until postnatal day (P) 12, then the normal increase in fiber density ceases, in some areas even distinctly decreases. This overlaps with activation of microglia, indicating an inflammatory and/or degenerative process. Here we studied, by in situ hybridization and immunohistochemistry (IHC), the expression of major histocompatibility complex (MHC) class I-related molecules and markers for cellular reactivity in hypothalamus of anx/anx mice. MHC class I transcript and -related proteins were found in arcuate nucleus (Arc), presumably both in neurons and glia, the latter also in areas innervated by AGRP (NPY) neurons. In the anx/anx hypothalamus, using TUNEL labeling, significantly higher number of apoptotic cells were found compared with +/+ mice, and active caspase 6 immunoreactivity was detected in degenerating NPY-fibers as well as signs of "microglia-associated cell death". In addition, Y1 receptor-labeled processes and soma of pro-opiomelanocortin (POMC) neurons, were markedly decreased at P21. These results support the hypothesis of degeneration of hypothalamic arcuate neuron populations in the anx/anx mice, whereby the AGRP system may be first affected, the changes in the POMC system being secondary in this process.

Classical major histocompatibility complex class I molecules in motoneurons: new actors at the neuromuscular junction.

Major histocompatibility complex (MHC) class I molecules have fundamental functions in the immune system. Recent studies have suggested that these molecules may also have non-immune functions in the nervous system, in particular related to synaptic function and plasticity. Because adult motoneurons express mRNAs for MHC class I molecules, we have examined their subcellular expression pattern in vivo and their role for the synaptic connectivity of these neurons. We observed immunoreactivity for classical MHC class I (Ia) protein in motoneuron somata, but the predominant expression was found in axons and presynaptically at neuromuscular junctions (NMJs). Peripheral nerve lesion induced a strong increase of motoneuron MHC class Ia (H2-K(b)/D(b)) mRNA, indicating a role for MHC class Ia molecules during regeneration. Accordingly, there was an accumulation of MHC class Ia proteins at the cut ends and in growth cones of motor axons after lesion. In K(b-/-)D(b-/-) mice (lacking MHC class Ia molecules), the time course for recovery of grip ability in reinnervated muscles was significantly delayed. Muscles from K(b-/-)D(b-/-) mice displayed an increased density and a disturbed distribution of NMJs and fewer terminal Schwann cells/NMJ compared with wild-type mice. A population of Schwann cells in sciatic nerves expressed the paired Ig receptor B, which binds to MHC class I molecules. These results provide the first evidence that neuronal MHC class Ia molecules are present in motor axons, that they are important for organization of NMJs and motor recovery after nerve lesion, and that their actions may be mediated via Schwann cells.

Altered expression of nectin-like adhesion molecules in the peripheral nerve after sciatic nerve transection.

Following axotomy several processes involving cell-cell interaction occur, such as loss of synapses, axon guidance, and remyelination. Two recently discovered families of cell-cell adhesion molecules, nectins and nectin-like molecules (necls) are involved in such processes in vitro and during development, but their roles in nerve injury have been largely unknown until recently. We have previously shown that axotomized motoneurons increase their expression of nectin-1 and nectin-3 and maintain a high expression of necl-1. We here investigate the expression of potential binding partners for motoneuron nectins and necls in the injured peripheral nerve. In situ hybridization (ISH) revealed a decreased signal for necl-1 mRNA in the injured nerve, whereas no signal for necl-2 was detected before or after injury. The signals for necl-4 and necl-5 mRNA both increased in the injured nerve and necl immunoreactivity displayed a close relation to axon and Schwann cell markers. Finally, signal for mRNA encoding necl-5 increased in axotomized spinal motoneurons. We conclude that peripheral axotomy results in altered expression of several necls in motoneurons and Schwann cells, suggesting involvement of the molecules in regeneration.

Integrin-laminin interactions controlling neurite outgrowth from adult DRG neurons in vitro.

A prerequisite for axon regeneration is the interaction between the growth cone and the extracellular matrix (ECM). Laminins are prominent constituents of ECM throughout the body, known to support axon growth in vitro and in vivo. The regenerative capacity of adult neurons is greatly diminished compared to embryonic or early postnatal neurons. Since most lesions in the nervous system occur in the adult, we have examined neurite outgrowth from adult mouse DRG neurons on four laminin isoforms (laminin-1/LM-111, laminin-2/LM-211, laminin-8/LM-411 and laminin-10/LM-511) in vitro. The growth on laminin-1 and -10 was trophic factor-independent and superior to the one on laminin-2 and -8, where growth was very poor in the absence of neurotrophins. Among other ECM proteins, laminins were by far the most active molecules. Using function-blocking antibodies to laminin-binding integrins, we identified non-overlapping functions of integrins alpha3beta1, alpha7beta1 and alpha6beta1 on different laminin isoforms, in that alpha3beta1 and alpha7beta1 integrins appeared to be specific receptors for both laminin-1 and-2, whereas integrin alpha6beta1 was a receptor for laminin-8 and-10. Lastly, by use of immunohistochemistry, expression of subunits of laminin-1, -2, -8 and -10 in sensory organs in the human epidermis could be demonstrated, supporting an important role for these laminins in relation to primary sensory axons.

MHC class I expression and synaptic plasticity after nerve lesion.

An axon lesion to a bulbar or spinal motoneuron is followed by a typical retrograde response at the cell body level, including the removal or 'stripping' of synapses from the perikaryon and dendrites of affected cells. Both activated microglia and astrocytes have been attributed roles in this process. The signalling pathways for this 'synaptic stripping' have so far been unknown, but recently a classical set of immune recognition molecules, the MHC class I molecules, have been shown to have a strong influence on the strength and pattern of the synapse elimination response. Thus, when MHC class I signalling is severely impaired in mice lacking the MHC class I subunit beta2-microglobulin (beta2m) and transporter associated with antigen processing 1 (TAP 1) genes, both of which are necessary for surface expression of MHC class I, there is a stronger elimination of synapses from injured neurons, with the surplus elimination directed towards clusters of putatively inhibitory synapses. Moreover, the regenerative capacity of motoneurons in such mice is lower than in wild-type animals. The expression of MHC class I, as well as MHC class I-related receptors in both neurons and glia, lend support to a hypothesis that classical immune recognition signalling between neurons and glia underlie part of the 'stripping' response.

The microglial networks of the brain and their role in neuronal network plasticity after lesion.

Microglia are the resident inflammatory cells of the central nervous system (CNS) extending a network of processes in the CNS parenchyma. Following axon lesion to neurons, most extensively studied in motoneurons, there is a typical retrograde response at the cell body level, including the removal or 'stripping' of synapses from the perikaryon and dendrites of affected cells. Microglia have been attributed a main and active role in this process, although also an involvement of activated astrocytes has been suggested. The signaling pathways for this 'synaptic stripping' have so far been unknown, but recently some classical immune recognition molecules, the MHC class I molecules, have been shown to have a strong influence on the strength and pattern of the synapse elimination response. Since there is an expression of MHC class I in both neurons and glia, in particular microglia, as well as MHC class I related receptors in axons and microglia, there are good reasons to believe that classical immune recognition signaling between neurons and glia underlies part of the 'stripping' response. A role for microglia in an interplay with synapses based on this type of signaling is further exemplified by the fact that, in the absence of some MHC class I related receptors normally found on microglia during development, profound effects on synaptic function and biochemistry have been demonstrated. Such effects may be linked to the development of various disorders of the CNS, such as degenerative disease.

Down-regulation of mRNAs for synaptic adhesion molecules neuroligin-2 and -3 and synCAM1 in spinal motoneurons after axotomy.

After peripheral axotomy, synapses are eliminated from the somata of spinal motoneurons. Recent evidence indicates that synaptic adhesion molecules play a role in maintenance of synaptic contacts, but so far such molecules have not been investigated in the context of synapse elimination after injury. In vitro, the neuroligins (NLGs) and SynCAM1 drive formation of synapses, and RNAi of NLGs results in decreased synaptic input, indicating an important role for these molecules in synaptic biology. To address potential involvement of NLGs and SynCAMs in postinjury synapse elimination, we investigated the mRNA expression of NLG1, -2, and -3; SynCAM1 and -3; and PSD-95--an intracellular NLG-binding scaffolding protein--in rat spinal motoneurons in control animals and after sciatic nerve transection (SNT). mRNA signals for NLG2, NLG3, SynCAM1, and SynCAM3, but not NLG1, were seen in uninjured motoneurons. Immunoreactivity for SynCAM was seen in close relation to synaptophysin immunoreactivity on the surface of motoneurons and in close relation to neurofilament immunoreactivity in the sciatic nerve. After axotomy, the signals for NLG2, NLG3, and SynCAM1 mRNAs decreased, whereas the signal for NLG1 mRNA remained undetectable and that for SynCAM3 remained at control levels. The signal for PSD-95 mRNA decreased gradually and reached approximately 50% of control values 2 weeks after axotomy. Thus the retrograde response to axotomy of spinal motoneurons involves a rapid down-regulation of NLG2, NLG3, and SynCAM1 mRNAs and a gradual decrease in PSD-95 mRNA. This indicates that down-regulation of synaptic adhesion molecules plays a role in postinjury synapse elimination.

Expression of Semaphorins, Neuropilins, VEGF, and Tenascins in Rat and Human Primary Sensory Neurons after a Dorsal Root Injury.

Dorsal root injury is a situation not expected to be followed by a strong regenerative growth, or growth of the injured axon into the central nervous system of the spinal cord, if the central axon of the dorsal root is injured but of strong regeneration if subjected to injury to the peripherally projecting axons. The clinical consequence of axonal injury is loss of sensation and may also lead to neuropathic pain. In this study, we have used in situ hybridization to examine the distribution of mRNAs for the neural guidance molecules semaphorin 3A (SEMA3A), semaphorin 3F (SEMA3F), and semaphorin 4F (SEMA4F), their receptors neuropilin 1 (NP1) and neuropilin 2 (NP2) but also for the neuropilin ligand vascular endothelial growth factor (VEGF) and Tenascin J1, an extracellular matrix molecule involved in axonal guidance, in rat dorsal root ganglia (DRG) after a unilateral dorsal rhizotomy (DRT) or sciatic nerve transcetion (SNT). The studied survival times were 1-365 days. The different forms of mRNAs were unevenly distributed between the different size classes of sensory nerve cells. The results show that mRNA for SEMA3A was diminished after trauma to the sensory nerve roots in rats. The SEMA3A receptor NP1, and SEMA3F receptor NP2, was significantly upregulated in the DRG neurons after DRT and SNT. SEMA4F was upregulated after a SNT. The expression of mRNA for VEGF in DRG neurons after DRT showed a significant upregulation that was high even a year after the injuries. These data suggest a role for the semaphorins, neuropilins, VEGF, and J1 in the reactions after dorsal root lesions.

Impeded interaction between Schwann cells and axons in the absence of laminin alpha4.

The Schwann cell basal lamina (BL) is required for normal myelination. Loss or mutations of BL constituents, such as laminin-2 (alpha2beta1gamma1), lead to severe neuropathic diseases affecting peripheral nerves. The function of the second known laminin present in Schwann cell BL, laminin-8 (alpha4beta1gamma1), is so far unknown. Here we show that absence of the laminin alpha4 chain, which distinguishes laminin-8 from laminin-2, leads to a disturbance in radial sorting, impaired myelination, and signs of ataxia and proprioceptive disturbances, whereas the axonal regenerative capacity is not influenced. In vitro studies show poor axon growth of spinal motoneurons on laminin-8, whereas it is extensive on laminin-2. Schwann cells, however, extend longer processes on laminin-8 than on laminin-2, and, in contrast to the interaction with laminin-2, solely use the integrin receptor alpha6beta1 in their interaction with laminin-8. Thus, laminin-2 and laminin-8 have different critical functions in peripheral nerves, mediated by different integrin receptors.

Integrin messenger RNAs in the red nucleus after axotomy and neurotrophic administration.

Integrins are cell surface receptors known to be important for regeneration in the peripheral nervous system. We have investigated the expression of integrin messenger RNAs in red nucleus neurons of adult rats after axotomy and administration of neurotrophic factors. Using radioactive in situ hybridization, messenger RNA for integrin subunits beta1, alpha3, alpha7 and alphaV could be detected. No change of any alpha subunit could be detected after axotomy. In contrast, a small upregulation of beta1 was detected after lesion. Administration of neurotrophin-3 induced a robust further increase in beta1 messenger RNA levels, whereas brain-derived neurotrophic factor did not. By analogy to the peripheral nervous system, we propose that integrins may be important for a regenerative response in central nervous system neurons.

On the Distribution of GAP-43 and its Relation to Serotonin in Adult Monkey and Cat Spinal Cord and Lower Brainstem.

By use of a monoclonal antibody, the distribution of growth-associated protein (GAP)-43-like immunoreactivity (LI) has been studied in the spinal cord of adult grey monkeys (Macaca fascicularis) and adult cats by use of immunofluorescence and peroxidase - antiperoxidase techniques. The brainstem was also studied with in situ hybridization histochemistry. In both monkeys and cats, a dense innervation of GAP-43-immunoreactive (IR) fibres was seen in close apposition to large cell bodies and their processes in the motor nucleus of the ventral horn. Double-labelling experiments revealed a high degree of coexistence between GAP-43- and 5-hydroxytryptamine (5-HT, serotonin)-LI in the monkey motor nucleus, while in the cat no such colocalization could be verified. At the electron microscopic level, GAP-43 labelling was seen as a coating of vesicles and axolemma inside the terminals. In both monkey and cat, cell bodies expressing mRNA encoding GAP-43 were demonstrated in the medullary midline raphe nuclei. A similar location was also encountered for mRNA for aromatic l-amino acid decarboxylase, an enzyme found in both catecholamine- and serotonin-containing neurons. The present results suggest that GAP-43 is present in the 5-HT bulbospinal pathway of the monkey. In the cat, GAP-43 mRNA-expressing cell bodies were demonstrated in areas where descending 5-HT neurons are located, but no convincing colocalization of 5-HT- and GAP-43-LI was found at spinal cord levels, despite the existence of extensive fibre networks containing either of the two compounds. Possible explanations for this species discrepancy are discussed. The function of GAP-43 in nerve terminals impinging on the motoneurons is unknown. However, it may play a role in transmitter release and/or plasticity, since such roles have been proposed for this protein in other systems.

Calcitonin Gene-related Peptide (CGRP)-like Immunoreactivity and CGRP mRNA in Rat Spinal Cord Motoneurons after Different Types of Lesions.

By use of the indirect immunofluorescence (IF) technique, radioimmunoassay (RIA) and in situ hybridization (ISH) histochemistry, the staining pattern, content and expression of calcitonin gene-related peptide (CGRP) in lumbar motoneurons of normal rats and rats subjected to sciatic nerve transection (SNT), ventral root transection (VRT), low thoracic spinal cord transection (SCT) alone or in combination with a subsequent SNT, as well as rats subjected to chemical lesioning of 5-hydroxytryptamine (5-HT) neurons by 5,7-dihydroxytryptamine (5,7-DHT), were studied. We here confirm that a large number of the lumbar motoneurons normally contain CGRP-like immunoreactivity (LI) and CGRP mRNA. SNT induced a transient increase in CGRP-LI, with a peak at days 2 - 5 after lesion, and normalized levels again after approximately 2 - 3 weeks. Comparable results were obtained with IF and RIA. This increase is probably a consequence of increased CGRP synthesis, since a parallel up-regulation of CGRP mRNA levels was seen. A normalization of CGRP mRNA did not occur during the period studied, despite an apparent normalization of peptide levels after 2 weeks, and this may in turn be due to an increased turnover and/or release of CGRP. The up-regulation of CGRP is probably caused by the axon injury itself, since a similar cellular reaction with respect to CGRP was observed in motoneurons subjected to VRT. However, SNT, which also lesions dorsal root afferents and causes a decline in CGRP-LI in the dorsal horn, induced an increase in CGRP-LI in motoneurons on the contralateral side also. Thus, it may be that severance of dorsal root afferents and/or changes in reflex activity may also influence the production of CGRP in motoneurons. SCT, which severs all descending synaptic input to the motor nucleus and causes a paralysis of muscles innervated by motoneurons below the lesion, resulted in a marked decline in both content of CGRP-LI (IF and RIA) and expression of CGRP mRNA. However, treatment with 5,7-DHT, which lesions 5-HT neurons, including those giving rise to the bulbospinal serotoninergic pathway, did not cause any dramatic changes in motor behaviour but induced an increase in both motoneuron content of CGRP-LI and expression of CGRP mRNA. In rats first subjected to SCT, which depresses CGRP, followed 2 weeks later by SNT, we found a marked increase in both content of CGRP-LI (IF and RIA) and expression of mRNA coding for CGRP. In summary our results show that the cellular production of the CGRP peptide, normally expressed in motoneurons, is influenced in a complex way by motoneuron injury as well as changes in the afferent input. There also appear to be important differences in the expression of CGRP in small (gamma) and large (alpha) motoneurons as well as between motoneurons of different nuclei, in normal as well as axotomized rats.

Neurotensin-like Peptides in the CNS of Lampreys: Chromatographic Characterization and Immunohistochemical Localization with Reference to Aminergic Markers.

Neurotensin (NT)-like peptides in the CNS of the lamprey Lampetra fluviatilis were studied by radioimmunoassay (C-terminal specific NT antiserum), reverse-phase HPLC and immunohistochemistry. Multiple peaks of NT-immunoreactive (-ir) material were observed upon HPLC, of which a major peak eluted in the position of bovine NT. Immunofluorescence histochemistry showed that a monoclonal antibody recognizing the N-terminal (1 - 11) fragment of NT, as well as two polyclonal NT antisera labelled a large number of cell bodies in the periventricular area of hypothalamus, including the postinfundibular commissural nucleus and the ventral and dorsal hypothalamic nuclei. Additional groups of NT-ir cells were observed in the preoptic nucleus, the postoptic commissural nucleus, the mesencephalic tegmentum (L.fluviatilis), and in the spinal cord (L.fluviatilis and Ichtyomyzon unicuspis). Dense NT-ir fibre plexuses were present in the caudal hypothalamus, corpus striatum, ventral mesencephalon, and in the dorsal horn and lateral margin of the spinal cord. At the ultrastructural level the lateral spinal margin showed NT-ir terminal structures, which in most cases were not associated with synaptic specializations, although occasional synaptic contacts with unlabelled elements were found. The relation between NT-ir and monoamine-containing cells was examined with immunofluorescence double-staining, using antisera to tyrosine hydroxylase (TH), 5-hydroxytryptamine (5-HT), and histamine respectively. In the periventricular nuclei of hypothalamus numerous TH-, 5-HT-, as well as histamine-ir cells were located in close association with NT-ir cells, but none of the aminergic markers could be detected within NT-ir neurons. The chemical properties as well as the anatomical distribution of lamprey NT-like peptides show several similarities with those present in mammals, suggesting that NT-containing neuronal systems in the CNS developed early in vertebrate phylogeny.

Large cholinergic nerve terminals on subsets of motoneurons and their relation to muscarinic receptor type 2.

The cholinergic C-bouton is a large nerve terminal found exclusively apposing motoneuron cell somata and proximal dendrites. The origin and function of the C-bouton is not known. An antiserum against the vesicular acetylcholine transporter was used to identify large cholinergic nerve terminals putatively of the C-type in close apposition to motoneuron cell somata. This type of nerve terminal was present in the rat spinal cord ventral horn, but only in some cranial motor nuclei. Fluoro-Gold tracing showed that subsets of spinal motoneuron cell somata were contacted by different numbers of putative C-boutons. Thus, motoneurons innervating an intrinsic foot muscle were contacted by about half the number of cholinergic terminals found on motoneurons of the predominantly fast-twitch gastrocnemius muscle. Slow-twitch soleus motoneurons showed an intermediate innervation. There was a strong correlation between the presence of putative C-boutons and muscarinic receptor 2 (m2)-like immunoreactivity (-LI) within a motor nucleus. By using confocal laser microscopy, the m2-LI appeared to be confined to the motoneuron cell membrane and strongly enriched beneath the C-type nerve terminal. Thus, our results suggested a differential distribution of large cholinergic C-boutons, depending on motoneuron type, and that the presence of this nerve terminal type is associated with m2-LI in the postsynaptic membrane.

Apoptosis of spinal interneurons induced by sciatic nerve axotomy in the neonatal rat is counteracted by nerve growth factor and ciliary neurotrophic factor.

We have previously shown that not only motoneurons and dorsal root ganglion cells but also small neurons, presumably interneurons in the spinal cord, may undergo apoptotic cell death as a result of neonatal peripheral nerve transection in the rat. With the aid of electron microscopy, we have here demonstrated that apoptosis in the spinal cord is confined to neurons and does not involve glial cells at the survival time studied (24 hours). To define the relative importance of the loss of a potential target (motoneuron) and a potential afferent input (dorsal root ganglion cell) for the induction of apoptosis in interneurons in this situation, we have compared the distributions and time courses for TUNEL labeling, which detects apoptotic cell nuclei, in the L5 segment of the spinal cord and the L5 dorsal root ganglion after sciatic nerve transection in the neonatal (P2) rat. In additional experiments, we studied the effects on TUNEL labeling of interneurons after treatment of the cut sciatic nerve with either ciliary neurotrophic factor (CNTF) to rescue motoneurons or nerve growth factor (NGF) to rescue dorsal root ganglion cells. The time courses of the TUNEL labeling in motoneurons and interneurons induced by the lesion show great similarities (peak at 8-48 hours postoperatively), whereas the labeling in dorsal root ganglion cells occurs later (24-72 hours). Both CNTF and NGF decrease the number of TUNEL-labeled interneurons, but there is a regional difference, in that CNTF preferentially saves interneurons in deep dorsal and ventral parts of the spinal cord, whereas the rescuing effects of NGF are seen mainly in the superficial dorsal horn. The results are interpreted as signs of a trophic dependence on both the target and the afferent input for the survival of interneurons neonatally.

Laminin chains in rat and human peripheral nerve: distribution and regulation during development and after axonal injury.

During nerve growth, axons are dependent upon contact with matrix components, such as laminins, for elongation, guidance, and trophic support. Semiquantitative in situ hybridization histochemistry and immunohistochemistry (IHC) were used to identify laminin chains in normal peripheral nerves, during postnatal development, after sciatic nerve transection (SNT), and after sciatic nerve crush (SNC). Laminin alpha2, alpha4, beta1, beta2, and gamma1 chain mRNAs were all expressed at high levels in newborn rat sciatic nerves with declining levels during later developmental stages. At the adult stage, no laminin chain mRNA was detectable. Of interest, the mRNA levels for alpha4 chain declined faster than those for alpha2. After SNT, laminin alpha2, alpha4, beta1, and gamma1 mRNA levels were up-regulated at the site of the injury, with the most profound reaction in the proximal nerve stump. Laminin alpha2 and alpha4 chains differed in that the mRNA levels of alpha4 were up-regulated earlier and declined quicker, whereas alpha2 had a later onset, with high levels remaining even after 6 weeks. After SNC, there was an initial up-regulation of the same laminin chain mRNAs as after SNT in the nerve, however, less intense, and at 6 weeks after SNC, all laminin mRNA levels studied had returned to normal. IHC of adult human normal and transected peripheral nerves stained positive for laminin alpha2, alpha4, beta1, and gamma1 chains in close relation to neurofilament labeled axons. Laminin alpha3, alpha4, alpha5, beta1, beta2, and gamma1 chains were found in blood vessel-like structures and alpha3, alpha4, alpha5, beta2, and gamma1 in the perineurium. These results and a previously published description of integrin regulation in spinal motoneurons suggest that both laminin-2 (alpha2beta1gamma1) and laminin-8 (alpha4beta1gamma1) are important for the postnatal nerve development and axonal regeneration after injury and that laminin-8 may have important functions especially early postnatally and early after adult nerve lesion.