Nitric oxide synthase expression and cell changes in dorsal root ganglia and spinal dorsal horn of developing and adult Rana esculenta indicate a role of nitric oxide in limb metamorphosis

Metamorphosis of amphibians requires reconfiguration of sensory and locomotor neural networks. In view of such plastic changes and implications of nitric oxide (NO) in neural developmental shaping, we examined via histochemistry and immunohistochemistry its synthetic enzyme nitric oxide synthase (NOS) in dorsal root ganglia (DRGs) and dorsal horn of the developing and adult frog Rana esculenta. In limb DRGs, NOS positivity was first and selectively detected just before limb bud appearance, increased during metamorphosis, and was then down-regulated. In adulthood, NOS was expressed in some DRG neurons at all segmental levels. Similar features were detected in the dorsal horn neuropil. In limb DRGs, cell counts in Nissl-stained sections revealed a twofold increase of differentiated neurons during metamorphosis and an additional twofold increase in adulthood. Perikaryal sizes in limb DRGs did not vary during metamorphosis but increased and were more heterogeneous in the adult frog, probably reflecting adaptation to body size. NOS and cell changes during metamorphosis were much less marked in DRGs at other levels. Carbocyanine tracing documented selective labeling of NOS-expressing hindlimb DRG neurons from the spinal nerve at the time of initiation of hindlimb movements. The findings show that, in limb DRG neurons, NOS parallels cell differentiation and limb development during metamorphosis. The data also provide evidence of NOS expression in DRG cells innervating the hindlimbs when sensorimotor circuits become functionally mature. This study indicates a key role of NO production in the maturation of sensory functions that subserves in amphibians the transition from swimming to tetrapod locomotion.     

Expression of Nitric-Oxide Synthase (NOS) in Injured CNS Neurons as Shown by NADPH Diaphorase Histochemistry

Recent studies have implicated nitric oxide (NO) in several mechanisms related to neuronal degeneration and synaptic plasticity. In the present study, two models of traumatic neuronal injury were used to examine the expression of NOS following neuronal injury and its relationship to axonal sprouting and neuronal degeneration. It was found that NOS is induced in a week of axonal injury in neurons that are normally NOS-negative. Spinal motoneurons express the enzyme after ventral root avulsion, but not after ventral root transection. Neurons of the nucleus dorsalis of the spinal cord express NOS after ipsilateral spinal cord hemisection. These two models provide information about the time course of NOS expression in injured neurons and the opportunity in future studies to determine the role of NOS and its product, NO, in CNS injury. Observations from the present study suggest that early NOS expression seems to be associated with axonal sprouting and growth. Interestingly, though, the neurons expressing lesion-induced NOS ultimately die. Whether NOS expression in these cells is related to their death is currently under investigation.     

Peripheral nerve graft and neurotrophic factors enhance neuronal survival and expression of nitric oxide synthase in Clarke's nucleus after hemisection of the spinal cord in adult rat.

The present study examined the effects of peripheral nerve (PN) graft and neurotrophic factors on the expression of nitric oxide synthase (NOS) and the survival of Clarke's nucleus (CN) neurons at the first lumbar spinal segment (L1) 15 days after hemisection of the spinal cord at T11. Normal intact CN neurons did not express NOS. Forty-one percent of the ipsilateral CN neurons survived after hemisection at T11, and 48% of the surviving neurons expressed NOS. Transplantation of PN graft at the lesion site promoted the survival of CN neurons to 71% and increased the expression of NOS to 70%. Continuous infusion of brain-derived neurotrophic factor, ciliary neurotrophic factor, and neurotrophic-3, but not glial cell-derived neurotrophic factor, at the lesion site enhanced the survival of CN neurons to about 65%. Among the surviving neurons about 70% were NOS-positive. These results indicated that transplantation of autologous PN graft or continuous infusion of neurotrophic factors could enhance the survival of axotomized CN neurons. In addition, the survival-promoting function of the neurotrophic agents was coincided with the upregulation of the expression of NOS. However, whether the upregulation of NOS expression in injured CN neurons is related to the rescue function or is a side effect of the neurotrophic factors is not clear and needed further investigation.     

大鼠脑片体外缺血状态纹状体中一氧化氮合成酶阳性神经元的时相变化及意义

无糖无氧人工脑脊液模拟脑缺血状态，用还原型尼克酰胺腺嘌呤二核苷酸脱氢酶反应观察大鼠离体脑片纹状体中一氧化氮合成酶阳性神经元的时相变化及神经损伤。结果显示：在缺血损伤严重的纹状体，一氧化氮合成酶阳性神经元缺血早期明显增多并深染，１ｈ后显著减少，提示纹状体的一氧化氮合成酶阳性神经元本身存在有限的抗损伤能力。     

Nitric oxide synthase in vagal sensory and sympathetic neurons innervating the guinea-pig trachea

Sympathetic (stellate and superior cervical ganglion) and sensory vagal (nodose and jugular ganglion) neurons innervating the guinea-pig trachea were labelled using a retrograde neuronal tracer (Fast Blue) and tested for immunoreactivity to nitric oxide synthase (NOS) and either tyrosine hydroxylase (TH; sympathetic ganglia) or substance P (SP; vagal afferent neurons). Approx. 3% of the sympathetic neurons innervating the trachea were NOS-positive. These neurons belonged to the non-catecholaminergic phenotype. Amongst the retrogradely labelled neurons in the vagal sensory ganglia, 5–10% of retrogradely labelled neurons in the nodose (inferior vagal) ganglion, and 10–20% of those in the jugular (superior vagal) ganglion were NOS-immunoreactive. All NOS-positive vagal afferent neurons labelled with retrograde tracer were negative for substance P. Accordingly, the results of these studies provide evidence that portions of the sympathetic and sensory innervation of the guinea-pig trachea is provided by NOS-immunoreactive neurons.     

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.     

NOS Expression in Nigral Cells after Excitotoxic and Non-excitotoxic Lesion of the Pedunculopontine Tegmental Nucleus

The substantia nigra (SN) receives afferents from cholinergic neurons of the pedunculopontine tegmental nucleus (PPTg), a neuronal population that shows high levels of nitric oxide synthase (NOS), the enzyme responsible for the synthesis of nitric oxide. We have investigated the effects of the injection in PPTg of two neurotoxins, kainic acid (an excitotoxic neurotoxin), and ethylcholine mustard azirinium ion (AF64A, a non-excitotoxic neurotoxin), upon the SN cells of the rat, by using choline acetyltransferase (ChAT) immunohistochemistry as a marker of cholinergic neurons, and nicotinamide adenine dinucleotide phosphate diaphorase (NADPHd) histochemistry and NOS immunohistochemistry as markers of nitric oxide-producing neurons. Our results show that in normal rats, the SN contains two populations of NOS-positive neurons: large cholinergic neurons of PPTg that invade the caudal region of the SN, and small elongated neurons lying in the SN pars compacta. After ipsilateral PPTg lesion, another population of nigral cells, constituted by medium sized neurons, became NADPHd/NOS-positive. This was much more evident in AF64A-injected rats, in which many medium sized neurons showed enzymatic activity and normal morphological features, at least during the 90 days after injection. Kainic acid-injected rats, in contrast, showed nigral cell degeneration, an effect not found in AF64A material, and only a few NOS-positive neurons. NADPHd/NOS activity was never present in degenerating neurons. These findings suggest that induction of NOS activity is not involved in nigral cell degeneration, and that nitric oxide could have a protective rather than a neurotoxic role. The possible role of nitric oxide in the pathogenesis of Parkinson's disease is discussed.     

Immunohistochemical localization of calbindin-D28k and calretinin in the spinal cord of Xenopus laevis

Immunohistochemical techniques were used to investigate the distribution and morphology of neurons containing the calcium-binding proteins calbindin-D28k (CB) and calretinin (CR) in the spinal cord of Xenopus laevis and determine the extent to which this organization is comparable to that of mammals. Most CB- and CR-containing neurons were located in the superficial dorsal gray field, but with distinct topography. The lateral, ventrolateral, and ventromedial fields also possessed abundant neurons labeled for either CB or CR. Double immunohistofluorescence demonstrated that a subpopulation of dorsal root ganglion cells and neurons in the dorsal and ventrolateral fields contained CB and CR. By means of a similar technique, a cell population in the dorsal field was doubly labeled only for CB and nitric oxide synthase (NOS), whereas in the ventrolateral field colocalization of NOS with CB and CR was found. Choline acetyltransferase immunohistochemistry revealed that a subpopulation of ventral horn neurons, including motoneurons, colocalized CB and CR. The involvement of CB- and CR-containing neurons in ascending spinal projections was demonstrated combining the retrograde transport of dextran amines and immunohistochemistry. Cells colocalizing the tracer and CB or CR were quite numerous, primarily in the dorsal and ventrolateral fields. Similar experiments demonstrated supraspinal projections from CB- and CR-containing cells in the brainstem and diencephalon. The distribution, projections, and colocalization with neurotransmitters of the neuronal systems containing CB and CR in Xenopus suggest that CB and CR are important neuromodulator substances with functions conserved in the spinal cord from amphibians through mammals.     

Calbindin-D28k and calretinin immunoreactivity in the spinal cord of the lizard Gekko gecko: Colocalization with choline acetyltransferase and nitric oxide synthase

The distribution of the calcium-binding proteins calbindin-D28k (CB) and calretinin (CR) was investigated in the spinal cord of the lizard Gekko gecko, by means of immunohistochemical techniques. Abundant cell bodies and fibers immunoreactive for either CB or CR were widely distributed throughout the spinal cord. Most neurons and fibers were labeled in the superficial dorsal horn, but numerous cells were also located in the intermediate gray and ventral horn. Distinct CB- and CR-containing cell populations were observed, although double immunohistochemistry revealed that 17-20% of the single-labeled cells for CB or CR in the dorsal horn contained both proteins. In addition, nitric oxide synthase was immunodetected in about 6% of the CB-positive neurons in the dorsal horn and in 10% in the ventral horn, whereas nitric oxide synthase was present in 9-13% of CR-positive cells in the dorsal horn and in 14% in the ventral horn. These doubly immunoreactive cells were restricted to areas IV, VII and VIII. Similar colocalization experiments revealed that 18-24% of the cholinergic cells in the ventral horn contained CB and 21-30% CR, with some variations throughout the length of the spinal cord. The pattern of distribution for CB and CR immunoreactivity in the spinal cord of the lizard, reported in the present study, is largely comparable to those reported for mammals, birds and anuran amphibians suggesting a high degree of conservation of the spinal systems modulated by these calcium-binding proteins.     

Nitric oxide synthase-positive neurons in the rat superior colliculus: colocalization of NOS with NMDAR1 glutamate receptor, GABA, and parvalbumin

We analyzed the potential input and output components of nitric oxide synthase (NOS)-containing neurons in the rat superior colliculus (SC). To identify whether NOS-positive neurons receive glutamatergic input we investigated the colocalization of NOS with NMDA receptor subunit R1 (NMDAR1). In addition, to examine whether putative nitric oxide synthesizing neurons represent a neurochemically specific or distinct subpopulation of cells in the SC we studied the colocalization of NOS with the neurotransmitter GABA, the calcium-binding proteins parvalbumin, calbindin and calretinin and with neuropeptides such as somatostatin, substance P and neuropeptide Y. We found that 90% of NOS-positive neurons in the superficial layers of the rat SC express NMDAR1. Nearly 20% of the population of nitridergic neurons also expresses GABA and 15% of them express parvalbumin. NOS-positive neurons in the superior colliculus did not contain calretinin, calbindin or either of the neuropeptides tested. The results of this study show that the capacity for synthesizing NO in the SC is largely restricted to neurons that receive glutamatergic inputs and that some of these neurons express GABA or parvalbumin.     

Colocalization of nitric oxide synthase and monoamines in neurons of the amphibian brain

By means of double immunohistofluorescence techniques, we have investigated the colocalization of nitric oxide synthase and tyrosine hydroxylase (TH) or serotonin (5-HT) in the central nervous system of the anurans Rana perezi and Xenopus laevis and the urodele Pleurodeles waltl. A wide codistribution of neuronal populations, expressing these markers, was found throughout the brain and spinal cord. In contrast, colocalization of these markers was rather restricted. Only in the caudal portion of the brainstem raphe column in anurans, approximately 80% of the 5-HT-positive cells were also NOS-immunoreactive, whereas in the urodele brain, about 40% of the serotonergic cells at the level of the glossopharyngeal motor nucleus were simultaneously NOS-positive. In various brain regions, a wide codistribution of NOS- and TH-containing neurons was observed, but real colocalization of nitrergic and catecholaminergic cells was only found in a small neuron population in the posterior tubercle of anuran amphibians. Therefore, in amphibians, only a distinct and small cell population within the serotonergic raphe column (anurans and urodele) and in the catecholaminergic posterior tubercle (anurans) seem to produce simultaneously nitric oxide.     

Development of interneurons with ipsilateral projections in embryonic rat spinal cord

Considerable progress has been made in recent years in identifying molecules with restricted expression in mammalian spinal cord at early developmental stages. However, the significance of the different expression patterns for most of these molecules is nuclear because so little is known about the development of various classes of spinal interneurons. Recently, we have characterized the development of rat spinal cord interneurons with an axon that crosses in the ventral commissure (Silos-Santiago and Snider, J. Comp. Neurol., 325:514, 1992). In the current study, we describe the morphological development of ipsilaterally projecting spinal interneurons in laminae V–VIII of the thoracic spinal cord. These neurons were labelled by retrograde lateral diffusion of DiI after crystals were placed in various locations in the embryonic thoracic cord. By E14, approximately 48 hours after the first interneurons are generated, eight different groups of ipsilateral interneurons are present in the spinal cord. By E15, these groups of ipsilateral interneurons have reached distinct locations within the gray matter. Even at this early stage, different groups of cells have elaborated characteristic dendritic arborizations. By E19, at least 17 different types of ipsilateral interneurons can be identified on the basis of location and dendritic morphology. In general, ipsilateral interneurons are located more dorsally and laterally than commissural interneurons at all stages of embryonic development. Furthermore, in comparison with commissural neurons, fewer ipsilateral interneurons have dendritic arbors with a mediolateral orientation in the transverse plane. This work demonstrates that rat embryonic spinal cord contains a large number of morphologically distinct classes of interneurons that extend axons into the ipsilateral lateral funiculus. These neurons can be distinguished from commissural neurons on the basis of location and morphology. These results, taken together with those from our previous study, provide a framework for the localization of gene expression to different classes of spinal interneurons at early developmental stages. © 1994 Wiley-Liss, Inc.     

NADPH diaphorase in the spinal cord of rats.

To identify spinal neurons that may synthesize nitric oxide, cells and fibers histochemically stained for NADPH diaphorase (a nitric oxide synthase) were studied in the spinal cord of rats. The histochemical reaction gave an image similar to the best Golgi impregnations, staining cells down to their finest processes. Transverse, horizontal, and parasagittal 50 and 100 microns sections were used to follow dendritic and axonal arborizations of stained neurons. Major cell groups were identified in the superficial dorsal horn and around the central canal (at all spinal levels), and in the intermediolateral cell column (at thoracic and sacral levels). Scattered positive cells were also found in deeper dorsal horn, ventral horn, and white matter. In some cases, axons of cells in the dorsal horn could be traced into the white matter; many of these cells resembled neurons projecting to various supraspinal targets. Stained cells in the intermediolateral column, which sent their axons into the ventral root, were presumed to be preganglionic autonomic neurons. Dense plexes of fibers were stained in laminae I and II and in the intermediolateral column. A large number of NADPH diaphorase-positive neurons in the spinal cord appear to be involved in visceral regulation. Fibers of the intermediolateral system had a special relationship with vasculature, suggesting that nitric oxide may help to couple neural activity with regional blood flow in the spinal cord. The abundance of NADPH diaphorase-positive neurons and fibers in the superficial dorsal horn suggests that nitric oxide may also be involved in spinal sensory processing.     

Insulin-like growth factor-I counteracts bFGF-induced survival of nitric oxide synthase (NOS)-positive spinal cord neurons after target-lesion in vivo.

We have used nitric oxide synthase (NOS) histochemistry as a perikaryal viability marker to trace the retrograde reaction of spinal cord intermediolateral (IML) sympathoadrenal projection (SAP)-neurons to target-removal, i.e., selective adrenomedullectomy and local administration of either insulin-like growth factor-I (IGF-I), basic fibroblast growth factor (bFGF) or a combination of both. Counting of NOS-positive preganglionic spinal cord neurons 4 weeks post surgery indicated that more than 80% of stained neurons were lost from the IML-cell column. This percentage loss corresponds to the numerical loss of NOS-stained SAP-neurons labeled retrogradely with Fast-blue prior to adrenomedullectomy. Basic FGF-supplementation at the site of lesion resulted in maintenance of the majority of NOS-positive IML-neurons, a finding confirmed by the survival rate of Fast-blue prelabeled SAP-neurons. Thus, besides maintenance of the structural integrity of SAP-neurons, bFGF prevents loss of intracellular NOS-activity which may reflect unaltered cell metabolism (and function) of these neurons following target-removal in vivo. By contrast, IGF-I failed to alter the rate of disappearance of NOS-staining and labeling index of neurons within the IML-cell column postlesion, suggesting that IGF-I is not neurotrophic for SAP-neurons by itself. Combined treatment with both factors resulted in a more widespread loss of NOS-stained and Fast-blue-prelabeled SAP-neurons than registered after bFGF-only treatment. No co-trophic effect of bFGF and IGF-I was evident; rather, the pronounced bFGF-induced rescuing effect was significantly suppressed by exogenous IGF-I in vivo, supporting the idea that this or another molecule induced by the treatment enhances rather than prevents retrograde degeneration and neuronal death within the adult lesioned IML-adrenal pathway.     

Neurons expressing NADPH-diaphorase in the developing human spinal cord

The present study used nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase histochemistry to identify populations of neurons containing nitric oxide synthase and to describe their putative migration during development of the human spinal cord. As early as week 6 (W6) of gestation, diaphorase expression was observed in sympathetic preganglionic neurons (SPNs) and interneurons of the ventral horn. As development proceeded, the SPNs translocated dorsally to form the intermediolateral nucleus, and the interneurons remained scattered throughout the ventral horn. In addition to the dorsal translocation of SPNs, a unique dorsomedially directed migratory pathway was observed. At later stages of development, other groups of SPNs were identified laterally in the lateral funiculus and medially in the intercalated and central autonomic regions. In addition, two "U-shaped" groups of diaphorase-labeled cells were identified around the ventral ventricular zone at W7. Cells of these groups appeared to translocate dorsally over the next weeks and presumably give rise to interneurons within the deep dorsal horn and surrounding the central canal. Furthermore, during W7-14 of gestation, the deep dorsal horn contained a number of diaphorase-positive cells, whereas the superficial dorsal horn was relatively free of staining. These data demonstrate that nitric oxide is present very early in human spinal cord development and that two unique cell migrations initially observed in rodents have now been identified in humans. Furthermore, nitric oxide may be expressed in some populations of neurons as they migrate to their final positions, suggesting that this molecule may play a role in neuronal development.     

Developmental expression of nitric oxide synthase in the rat diencephalon with special reference to the thalamic paratenial nucleus

Using immunohistochemistry and in situ hybridization the distribution of nitric oxide synthase (NOS) was investigated in the rat brain during pre- and postnatal development. At E15 weak NOS-like immunoreactivity (NOS-LI) could be seen in the differentiation field of the anterior hypothalamus. At E17 strong NOS-LI was observed in the developing neurons of the hypothalamic paraventricular nucleus, supraoptic nucleus, anterodorsal nucleus and lateral hypothalamic areas. In the thalamic paratenial nucleus a strong NOS-LI was observed in these neurons at E17, E18 and P1 with a weaker intensity at P3, P7, P9 and P15, whereas at P30 and in adult rats no NOS-positive neurons could be detected. NOS expression at E17 and P3 was verified by in situ hybridization. These results suggest that NO may have a developmental role at least in one of the regions studied, the thalamic paratenial nucleus.     

Variability in the occurrence of nitric oxide synthase immunoreactivity in different populations of rat sympathetic preganglionic neurons

The proportion of sympathetic preganglionic neurons (SPN) showing nitric oxide synthase (NOS) immunoreactivity appears to vary with innervation target and blood pressure level. For normotensive Sprague‐Dawley rats (SD), we evaluated peroxidase immunolabelling for choline acetyltransferase (ChAT) plus NOS in spinal cord segments T1–L2 and assessed NOS immunofluorescence in SPN retrogradely labelled with cholera toxin B subunit from the adrenal medulla (AM) or superior cervical (SCG), coeliac (CG), or major pelvic (MPG) ganglia. We also compared the distributions and numbers of NOS‐positive and NOS‐negative/ChAT‐positive lateral horn neurons in SD with those in normotensive Wistar‐Kyoto (WKY) and spontaneously hypertensive rats (SHR). In SD, WKY, and SHR, rostrocaudal, dorsoventral, and mediolateral differences occurred in the distributions of NOS‐positive and NOS‐negative/ChAT‐positive neurons in the intermediolateral cell column (IML), whereas the two groups were similarly distributed throughout the central autonomic area (CAA). Among the four retrogradely labelled populations of SPN, the percentages showing NOS immunoreactivity differed (CG‐projecting, 54.8% ± 0.7%; SCG‐projecting, 75.3% ± 1.2%; MPG‐projecting, 89% ± 1.1% and AM‐projecting, 98.6% ± 0.2%). Within each retrogradely labelled group of SPN, the NOS‐positive proportion also varied with subnuclear location (e.g., 25.5% ± 4.0% of CG‐projecting SPN in the CAA vs. 82.7% ± 7.6% of CG‐projecting SPN in the dorsolateral funiculus). The numbers of NOS‐positive and NOS‐negative/ChAT‐positive neurons in T9–T11 were the same in SD and SHR but differed in WKY. Our results show that the expression of NOS within SPN varies depending on the target that they innervate and also on their subnuclear location. Our data indicate that there are no anatomical differences between nitric oxide‐synthesizing SPN in normotensive SD and hypertensive SHR. J. Comp. Neurol. 514:492–506, 2009. © 2009 Wiley‐Liss, Inc.     

Acetylcholinesterase Immunolesioning: Regional Vulnerability of Preganglionic Sympathetic Neurons in Rat Spinal Cord

Rats given antibodies against acetylcholinesterase (AChE) develop sympathetic dysfunction stemming from losses of preganglionic neurons in spinal cord. Central effects of AChE antibodies are surprising since IgG does not readily cross the blood–brain barrier, and lesions of peripheral terminals should not cause cell death. This study was designed to explore the distribution of central neural damage and to investigate features that might account for vulnerability. Rat spinal cord and brainstem were stained for choline acetyltransferase (ChAT) and nitric oxide synthase (NOS) immunoreactivity. Four months after administration of AChE antibodies, ChAT-positive neurons in the intermediolateral nucleus (IML) were 61–66% fewer throughout the thoracolumbar cord (T1, T2, T8, T12, L1). NOS-positive neurons in these loci were affected to the same extent by antibody-treatment, although they were only two-thirds as numerous. By contrast, neurons in the central autonomic nucleus of the thoracolumbar cord were scarcely affected. These results point to immunochemical differences in the central autonomic outflow, which may partially explain the puzzling selectivity of neural damage in AChE immunolesioning. Different results were obtained after guanethidine sympathectomy, which ablated nearly all neurons in the superior cervical ganglion without any effect on preganglionic neurons in the IML. Therefore, if the central effects of antibodies are indirectly mediated by loss of trophic support from the periphery, this support cannot arise from adrenergic neurons but must come from other ganglionic cells.     

Transient ischemia-induced changes of excitatory amino acid carrier 1 in the ventral horn of the lumbar spinal cord in rabbits

OBJECTIVES: To examine temporal changes of EAAC1 immunoreactivity and its protein level in the spinal ventral horn after transient ischemia in the rabbit to investigate the correlation between neuronal cell death and EAAC1 in the ventral horn of spinal cord. METHODS: White rabbits weighing 2.5-3.0 kg were anesthetized with a mixture of 2.5% isoflurane in 30% oxygen and 70% nitrous oxide, and the abdominal aortic artery below the left renal artery was occluded for 15 minutes. At designated times after reperfusion, the immunohistochemical and Western blot analysis for EAAC1 was conducted using tissues of the seventh lumbar spinal segment. RESULTS: EAAC1 immunoreactivity was detected in the neurons of the normal spinal cord. EAAC1 immunoreactivity and protein level reduced significantly 30 minutes after ischemia/reperfusion, but EAAC1 immunoreactivity and protein level again increased by 80% versus sham 3 hours after ischemia. At this time point, neurological defect in hindlimb was also detected. Thereafter, EAAC1 immunoreactivity and protein levels remained to be attenuated in the ventral horn of spinal cord until 48 hours after ischemia. CONCLUSION: The significant change in EAAC1 expression and motor defects at early time after transient spinal cord ischemia relates to the acute events following ischemia/reperfusion. These results indicate that EAAC1 has an important role in the modulation of glutamate homeostasis in ischemic neurons in the spinal ventral horn.     

Nicotine attenuates arachidonic acid-induced overexpression of nitric oxide synthase in cultured spinal cord neurons

Primary spinal cord trauma can initiate a cascade of pathophysiologic events which markedly contribute to the expansion and amplification of the primary insult. The detailed mechanisms of these secondary neurochemical reactions are largely unknown; however, they involve membrane lipid derangements with the release of free fatty acids, in particular, arachidonic acid (AA). AA can induce several injury effects on spinal cord neurons. We hypothesize that upregulation of nitric oxide synthase (NOS) is among the most important mechanisms of arachidonic-acid-induced neuronal dysfunction and that nicotine can attenuate this effect. To study these hypotheses, spinal cord neurons were exposed to AA and/or nicotine, and several markers of neuronal nitric oxide synthase (nNOS) metabolism were measured. In addition, cotreatments with either inhibitors of nicotinic receptors or inhibitors of specific NOS isoforms were employed. Treatment with AA markedly increased activity of nNOS, as well as mRNA and protein levels of this enzyme. Changes in nNOS expression were accompanied by an increase in cellular cGMP and medium nitrite levels. Pretreatment with nicotine decreased AA-induced overexpression of nNOS and elevation of nitrite levels. In addition, it appeared that these nicotine effects could be partially modulated both by the alpha7 nicotinic receptors or by nonreceptor mechanisms. Alternatively, the observed changes could also be mediated by an alternate nicotinic receptor mechanism which is not blocked by alpha-bungarotoxin or mecamylamine. Results of the present study indicate that exposure to AA can lead to induction of nNOS in cultured spinal cord neurons. In addition, nicotine can exert a neuroprotective effect by attenuation of AA-induced upregulation of nNOS metabolism. These data may have therapeutic implications for the treatment of acute spinal cord trauma.