Triphosphopyridine nucleotide-dependent enzymes in the developing spinal cord of the rabbit

Abstract— Total lipid and the activity of five enzymes closely related to the generation of NADPH have been measured in the anterior horn region and dorsal columns of rabbit spinal cord during the period of rapid myelination. Lipid deposition progressed to a much greater extent in the dorsal columns than in the anterior horn region; however, the age at which one-half of the total adult level of lipid accumulated in both regions was the same, i.e. 19-20 days after birth. During the first 15 days of postnatal development of the dorsal columns, glucose-6-phosphate dehydrogenase changed in parallel with lipid content; however, in the anterior horn region changes in lipid were not accompanied by increases in glucose-6-phosphate dehydrogenase. In contrast to changes in glucose-6-phosphate dehydrogenase, the activity of malic enzyme increased in the anterior horn region but remained relatively constant in the dorsal columns during development. The activities of two other enzymes of the pentose phosphate pathway, 6-phosphogluconate dehydrogenase and transketolase, measured at various intervals after birth, did not directly parallel changes in the activity of glucose-6-phosphate dehydrogenase in the dorsal columns. In both areas of the developing spinal cord the activity of NADP⁺-dependent isocitrate dehydrogenase was greater than the activities of the other three dehydrogenases but it did not parallel changes in lipid content of either region. A relationship between the requirements for reducing equivalents and the activities of the four NADP⁺-dependent dehydrogenases is suggested by the finding that both areas of the adult spinal cord contained lower activities of these enzymes than those observed during the initial 26 days of development. The differences noted in the two areas of the spinal cord during development suggest that mechanisms for the generation of NADPH differ in gray and white matter.     

The quantitative histochemistry of multiple sclerosis plaques: acid proteinase and other acid hydrolases.

Abstract— Sensitive micromethods were used to study the plaques, adjacent white matter and remote, grossly normal white matter from two cases of multiple sclerosis and to compare them with white matter from normal controls. Lipid-free dry wt/unit of volume was found to be similar for plaques and for normal white matter, reflecting a high water content of plaque tissue and establishing a base for comparison of enzyme activities. Elevations of acid proteinase in and around plaques were confirmed, but they were far exceeded by the increases in acid phosphatase; other acid hydrolases (β-galactosidase, β-glucuronidase and dipeptidyl arylamidase II) showed no significant or consistent changes. However, an acid lipase-esterase hydrolysing 4-methylumbelliferyl oleate was about 30% as active in plaques as in normal-appearing white matter. Glucose-6-phosphate dehydrogenase was unchanged except in one plaque, but lactic dehydrogenase was markedly elevated both in plaques and adjacent white matter. The grossly normal white matter of MS patients, although histologically far from normal (showing gliosis, perivascular infiltrations and small plaques), did not differ significantly from controls with regard to the activity of any of the enzymes studied. DNA levels were much reduced in plaques, but comparisons were difficult because of the apparent gliosis in normal white matter. Decreases in dry wt/unit vol, reflecting partial demyelination, could be shown to extend in a gradient to a distance of about 2 mm. from the edge of certain plaques.     

Microdetermination of 2-Deoxyglucose and 2-Deoxyglucose 6-Phosphate to Determine Glucose Utilization Rates in Single Neurons and Small CNS Regions After Injecting Nontracer Amounts of 2-Deoxyglucose

A nontracer amount (0.25 mmol/kg of body weight) of 2-deoxyglucose (DG) was intravenously injected into rats, which were frozen 2 and 4 min later in liquid nitrogen. Freeze-dried samples of CNS regions and cell bodies of spinal motor neurons were prepared, and the concentrations of glucose, glucose 6-phosphate, DG, and DG 6-phosphate (DG6P) in them were microassayed after 3,000-1,500,000-fold amplification using an enzymatic amplification reaction, NADP cycling. Based on the time course of glucose, DG, and DG6P concentrations in arterial plasma and the anterior horn of the spinal cord, the Sokoloff-type rate equations for DG and DG6P concentrations were mathematically solved, and the resultant DG and DG6P concentration functions were fitted to the data points using the nonlinear least-squares fitting SALS package program. This fitting provided four rate constants for the functions and supported the theoretical basis for our calculations of glucose utilization rate (GUR) when DG was administered in nontracer amounts. The GUR was highest in the spinal motor neurons and lowest in the white matter of the cerebellum. Neuron-rich structures, such as the cerebellar molecular and granular layers and the anterior horn of the spinal cord, had higher GUR values than the white matter of the cerebellum and spinal cord.     

Gamma interferon is critical for neuronal viral clearance and protection in a susceptible mouse strain following early intracranial Theiler's murine encephalomyelitis virus infection

We evaluated the role of gamma interferon (IFN-gamma) in protecting neurons from virus-induced injury following central nervous system infection. IFN-gamma(-/-) and IFN-gamma(+/+) mice of the resistant major histocompatibility complex (MHC) H-2(b) haplotype and intracerebrally infected with Theiler's murine encephalomyelitis virus (TMEV) cleared virus infection from anterior horn cell neurons. IFN-gamma(+/+) H-2(b) mice also cleared virus from the spinal cord white matter, whereas IFN-gamma(-/-) H-2(b) mice developed viral persistence in glial cells of the white matter and exhibited associated spinal cord demyelination. In contrast, infection of IFN-gamma(-/-) mice of the susceptible H-2(q) haplotype resulted in frequent deaths and severe neurologic deficits within 16 days of infection compared to the results obtained for controls. Morphologic analysis demonstrated severe injury to spinal cord neurons in IFN-gamma(-/-) H-2(q) mice during early infection. More virus RNA was detected in the brain and spinal cord of IFN-gamma(-/-) H-2(q) mice than in those of control mice at 14 and 21 days after TMEV infection. Virus antigen was localized predominantly to anterior horn cells in infected IFN-gamma(-/-) H-2(q) mice. IFN-gamma deletion did not affect the humoral response directed against the virus. However, the level of expression of CD4, CD8, class I MHC, or class II MHC in the central nervous system of IFN-gamma(-/-) H-2(q) mice was lower than those in IFN-gamma(+/+) H-2(q) mice. Finally, in vitro analysis of virus-induced death in NSC34 cells and spinal motor neurons showed that IFN-gamma exerted a neuroprotective effect in the absence of other aspects of the immune response. These data support the hypothesis that IFN-gamma plays a critical role in protecting spinal cord neurons from persistent infection and death.     

Zur Chemodifferenzierung des Rückenmarks und der Spinalganglien der Ratte

Zusammenfassung Alkalische Phosphatase, saure Phosphatase, Glukose-6-Phosphatdehydrogenase und NADH-Diaphorase können erstmalig am 13., unspezifische Esterase am 15. Embryonaltag (ET) im Cytoplasma der Neuroblasten des Vorderhorns und der Spinalganglien nachgewiesen werden. Ein Unterschied zwischen zervikalem und lumbalem Teil des Rückenmarks besteht nicht. Während der weiteren Entwicklung breiten sich die Enzymreaktionen in der grauen Substanz nach dorsal aus. Am Ende der Tragzeit entspricht die Verteilung der Fermente der erwachsener Tiere. — Die Azetylcholinesterase reagiert ab 14. ET bis zur Geburt in den Hintersträngen stark positiv und ab 15. ET gleichzeitig in den Vorderhornzellen und Spinalganglien. Nach der Geburt sind die Perikarya der Vorderhornzellen Azetylcholinesterase-frei, dafür reagiert die Zelloberfläche positiv. — Die lysosomale Lokalisation der sauren Phosphatase in den Vorderhornzellen kann sehr früh (15. ET) nachgewiesen werden. Glukose-6-Phosphatdehydrogenase und NADH reagieren in diesen Zellen während der Embryonalzeit diffus. Ab 18. ET reagiert in der grauen Substanz das Scitenhorn bei Nachweis der Glukose-6-Phosphatdehydrogenase und NADH am kräftigsten. — Das Ependym und die Commissura anterior besitzen vom 13. ET bis zur Geburt eine deutliche positive Reaktion für saure Phosphatase, Glukose-6-Phosphatdehydrogenase und NADH. Gliazellen haben während der Embryonalentwicklung keine nachweisbare Enzymaktivität. Diese tritt erstmalig für Glukose-6-Phospahtdehydrogenase und NADH am 1. Lebenstag auf und steigert sich abhängig vom Fortschreiten der Myelinisation. — Die Neurone im Spinalganglion zeigen unterschiedliche Fermentreaktionen, wahrscheinlich als Ausdruck verschiedener Zellaktivität. — Belastung durch Schwimmen zieht keine Veränderungen der Enzymaktivitäten im Rückenmark und Spinalganglion nach sich.

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Chemodifferentiation of the spinal cord and spinal ganglion of the rat

Summary In the cytoplasm of neuroblasts of ventral horn and spinal ganglia alkaline phosphatase, acid phosphatase, glucose-6-phosphate dehydrogenase and NADH diaphorase can be first demonstrated on the 13th embryonic day and non-specific esterase activity on the 15th embryonic day. There are no differences between the cervical and the lumbal spinal cord. During the further development the enzyme activities in the gray matter extend in a dorsal direction. At the end of pregnancy the distribution of enzymes is like that in adult animals. — The acetylcholinesterase reaction is strongly positiv in the posterior column from the 14th embryonic day to birth, and from the 15th embryonic day onward also in the nerve cells of the ventral horn and spinal ganglia. After birth the pericarya of the ventral horn are devoid of acetylcholinesterase. There is, however, a positive reaction on the surface of the cells. — The lyososomal localization of acid phosphatase can be demonstrated on the 15th embryonic day in the nerve cells of ventral horn. Glucose-6-phosphate dehydrogenase and NADH diaphorase exhibit a diffuse reaction in these cells during embryonic life. From the 18th embryonic day onward the lateral horn of the gray matter shows the highest activities of glucose-6-phosphate dehydrogenase and NADH-diaphorase. — In the ependyma and the anterior commissure acid phosphatase, glucose-6-phosphate dehydrogenase and NADH diaphorase can be visualized from the 13th embryonic day to birth. — In glial cells no enzymes can be demonstrated during embryonic life. On the 1st day after birth glucose-6-phosphate dehydrogenase and NADH diaphorase occur. These enzyme activities then increase depending on the degree of myelination. — In the neurons of spinal ganglia the enzyme reactions show marked differences probably indicating functional differences. — Continuous swimming does not lead to demonstrable enzyme changes in spinal cord and spinal ganglia.

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Stipendiat des Deutschen Akademischen Austauschdienstes.     

Growth factor receptors in amyotrophic lateral sclerosis

The regional distribution of nerve growth factor (NGF) and insulin-like growth factor-1 (IGF-1) receptors in human spinal cords from controls and amyotrophic lateral sclerosis (ALS) patients was studied by quantitative autoradiography. High-affinity nerve growth factor receptors were found to be distributed to a similar extent within the various segments of the human spinal cord and predominantly within the substantia gelatinosa of the dorsal horn, whereas no significant binding could be detected in the motor-neuron areas. A similar pattern of binding was obtained in the ALS spinal cords. Moreover, no reexpression of NGF receptors could be demonstrated in the motor-neuron areas of ALS spinal cords. When comparing¹²⁵I-IGF-1 binding in the different spinal levels of normal spinal cord, the same distribution pattern was found in which the binding was highest in the central canal > dorsal horn > ventral horn > white matter. In the ALS cases, although a general upregulation of IGF-1 receptors was observed throughout the spinal cord, significant increases were observed in the cervical and sacral segments compared to controls. The cartography of IGF-1 receptors in the normal spinal cord as well as the change of these receptors in diseased spinal cord may be of importance in future treatment strategies of ALS.     

Nonhematopoietic erythropoietin derivatives prevent motoneuron degeneration in vitro and in vivo

Chronic treatment with asialo erythropoietin (ASIALO-EPO) or carbamylated erythropoietin (CEPO) improved motor behavior and reduced motoneuron loss and astrocyte and microglia activation in the cervical spinal cord of wobbler mice, an animal model of amyotrophic lateral sclerosis, but had no effect on hematocrit values. ASIALO-EPO and CEPO, like the parent compound EPO, protected primary motoneuron cultures from kainate-induced death in vitro. Both EPO receptor and the common CD131 beta chain were expressed in cultured motoneurons and in the anterior horn of wobbler mice spinal cord. Our results strongly support a role for the common beta chain CD131 in the protective effect of EPO derivatives on motoneuron degeneration. Thus CEPO, which does not bind to the classical homodimeric EPO receptor and is devoid of hematopoietic activity, could be effective in chronic treatment aimed at reducing motoneuron degeneration.     

The 21-Aminosteroid U-74389F Attenuates Hyperexpression of GAP-43 and NADPH-Diaphorase in the Spinal Cord of Wobbler Mouse,a Model for Amyotrophic Lateral Sclerosis

The wobbler mouse suffers an autosomal recessive mutation producing severe neurodegeneration and astrogliosis in spinal cord. It has been considered a model for amyotrophic lateral sclerosis. We have studied in these animals the expression of two proteins, the growth-associated protein (GAP-43) and the NADPH-diaphorase, the nitric oxide synthesizing enzyme, employing immunocytochemistry and histochemistry. We found higher expression of GAP-43 immunoreactivity in dorsal horn, Lamina X, corticospinal tract and ventral horn motoneurons in wobbler mice compared to controls. Weak NADPH-diaphorase activity was present in control motoneurons, in contrast to intense labeling of the wobbler group. No differences in diaphorase activity was measured in the rest of the spinal cord between control and mutant mice. A group of animals received subcutaneously for 4 days a 50 mg pellet of U-74389F, a glucocorticoid-derived 21-aminosteroid with antioxidant properties but without glucocorticoid activity. U-74389F slightly attenuated GAP-43 immunostaining in dorsal regions of the spinal cord from wobblers but not in controls. However, in motoneurons of wobbler mice number of GAP-43 immunopositive neurons, cell processes and reaction intensity were reduced by U-74389F. The aminosteroid reduced by 50% motoneuron NADPH-diaphorase activity. Hyperexpression of GAP-43 immunoreactivity in wobbler mice may represent an exaggerated neuronal response to advancing degeneration or muscle denervation. It may also be linked to increased nitric oxide levels. U-74389F may stop neurodegeneration and/or increase muscle trophism and stop oxidative stress, consequently GAP-43 hyperexpression was attenuated. Wobbler mice may be important models to evaluate the use of antioxidant steroid therapy with a view to its use in human motoneuron disease.     

扬子鳄颈髓一氧化氮合酶(NOS)和乙酰胆碱酯酶(AChE)阳性神经元的分布

本实验分别应用还原型尼克酰胺嘌呤二核苷酸脱氢酶（ＮＡＤＰＨ－ｄ）和乙酰胆碱酯酶（ＡＣｈＥ）方法,对扬子鳄颈髓ＮＯＳ和ＡＣｈＥ阳性神经元的分布进行了研究。结果表明：颈髓前角、中央灰质均含有ＮＯＳ和ＡＣｈＥ阳性神经元,颈髓后角有较为丰富的ＮＯＳ和ＡＣｈＥ阳性纤维和终末以及显色淡的ＮＯＳ阳性神经元。     

Immunohistochemical investigations of excitotoxicity in experimental spinal cord trauma

The concept of "excitotoxicity" assumes that high concentration of glutamate (main excitatory neuromediator) acting through specific receptors leads to damage of cells due to an influx of calcium ions. Proteins called "excitatory amino acid transporters" (EAATs), present in astroglia, play important role in the removal of glutamate. We investigated the expression of GluR2 (glutamate receptor), EAAT1, and EAAT2 by immunohistochemistry in formalin-fixed, paraffin-embedded rat spinal cords, previously subjected to experimental mechanical trauma. In the injured spinal cords, an elevated immunoreactivity of GluR2 was noted even 10 min after trauma and was still observed 2 days after injury. Strong immunoreactivity was observed not only in many cells in gray matter but also in some cells in white matter (probably glial cells). In the injured spinal cords, we observed stronger (as compared with controls) expression of EAATs in the white matter, especially 6 hours after injury. The results support the role of excitotoxicity in mechanical trauma of spinal cord suggesting a possibility of long lasting elevated expression of glutamate receptor. It may help to understand and to explain beneficial action of "anti-glutamate" drugs, reported by other investigators.     

Astrocyte proliferation induced by wobbler astrocyte conditioned medium is blocked by tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) neutralizing antibodies in vitro.

The wobbler mutant mouse (wr/wr) displays motoneuron degeneration and astrocyte reactivity in the spinal cord. We have previously reported that, in vitro, primary wobbler astrocytes display morphological and biochemical changes. In this report, we show that wobbler astrocyte conditioned medium enhances the in vitro proliferation of normal neonatal primary astrocytes. This stimulated proliferation is correlated with high levels of IL1-beta and TNF-alpha cytokines in the conditioned medium of wobbler astrocytes. Neutralizing antibodies directed against both IL1-beta and TNF-alpha block the wobbler astrocyte conditioned medium-enhanced astrocyte proliferation. Moreover, IL1-beta and TNF-alpha mRNAs are elevated in the wobbler spinal cord. All these data suggest that diffusible IL1-beta and TNF-alpha are involved in the processus of astrogliosis observed in the wobbler spinal cord.     

Raised Thyrotrophin-Releasing Hormone, Pyroglutamylamino Peptidase, and Proline Endopeptidase Are Present in the Spinal Cord of Wobbler Mice but Not in Human Motor Neurone Disease

The Wobbler mouse (wr) is a mutant that exhibits loss of anterior horn cells in the spinal cord and brainstem and subsequent muscle wasting, particularly of the forelimbs and neck. The wr mice, 2-3 months of age, were found to have increased levels of immunoreactive-thyrotrophin-releasing hormone (ir-TRH) in the spinal cord and pons and medulla, but not in other CNS areas. This increase was observed in dorsal and ventral cord and at cervical, thoracic, and lumbar levels and was confirmed by HPLC to be authentic TRH. The levels of immunoreactive-somatostatin, -neurotensin, and -substance P were not raised in the CNS of wr mice. The activities of two peptidases capable of degrading TRH, pyroglutamylaminopeptidase (PGAP, EC 3.4.11.8) and proline endopeptidase (PEP, EC 3.4.21.26), and the level of 5-hydroxyindoleacetic acid were also raised in the spinal cord of 2-3-month-old wr mice although the activities of alanine aminopeptidase and lactate dehydrogenase and the level of 5-hydroxytryptamine were not. Increased spinal cord levels of ir-TRH and PGAP and PEP activities were not observed in the 1-month-old wr mice. In addition, a pilot study using spinal cord obtained at autopsy from three patients with motor neurone disease and 12 control subjects indicated no increase in spinal cord ir-TRH, PGAP, or PEP in human motor neurone disease.     

Time of origin of cells in the histiogenesis of the spinal cord]

In order to establish the moment of appearance of neuroblasts and ectoglia of the spinal cord the autoradiographic study with the use of H3-thymidine and C14-thmidine injected to pregnant mice with the intervals between injections 121/2 or 24 hours was undertaken. It was establised that spinal neurons were removed from the nervous tube beginning from the 10th up to 13th days of embroyogenesis. The motoneurons of the anterior horn were the first to appear (10th-12th days), the neurons of the intermideate zone were the next to appear (11th - 12th days) and the last were the neurons of posterior horn (13th day). Beginning from the 13th day of embryogenesis there appeared the ectoglia which migrated following meurblasts two days later. The saturation of the grey matter with glial cells and the saturation of the white matter with Schwann cells was brought about by means of additional multiplication at the site of the glioblasts removed from the nervous tube. The main function of the matrix layer neuroepithelium of the nervous tube as a provider of cells to the spinal cord terminated on the 15th day of embryogenesis.     

The post-mortem stability of certain oxidative enzymes in brain and spinal cord

Summary An investigation has been carried out on the stability of several enzymes in portions of rabbit brain and spinal cord kept at controlled temperatures between 22 and 37° C for periods up to 24 hours before processing for enzyme activity. The enzymes studied were NAD diaphorase, succinate, lactate, glutamate and glucose-6-phosphate dehydrogenases, and monoamine oxidase. One-wavelength plug cytophotometric measurements of enzyme activity were carried out on Purkinje cells, neuropil of the granular layer of the cerebellar cortex and on anterior horn cells.Succinate dehydrogenase activity proved to be stable after 24 hours post-mortem exposure at 37°C. Lactate dehydrogenase, NAD diaphorase and monoamine oxidase activities were less stable at the higher temperatures but were stable at 22°C. Glutamate and glucose-6-phosphate dehydrogenase activities fell significantly with exposure at 22°C. It thus appears possible to make valid histochemical measurements of the activities of certain oxidative enzymes in selected post-mortem brain material.This research was aided by a grant from the National Health and Medical Research Council of Australia.     

Role of VP2 amino acid 141 in tropism of Theiler's virus within the central nervous system.

Following intracranial inoculation, Theiler's virus causes either an acute encephalitis (strain GDVII) or a chronic demyelinating disease (strain DA). The DA strain sequentially infects the grey matter of the brain, the grey matter of the spinal cord, and, finally, the white matter of the spinal cord, where it persists in glial cells and causes demyelinating lesions. Analysis of the phenotype of recombinant viruses has shown that the viral capsid contains determinants for persistence and demyelination. Our previous studies showed that a Lys at position 141 of the VP2 capsid protein (VP2-141) could render a chimeric virus persistent. We also reported that another recombinant virus, virus R5, migrated from the grey matter of the brain to that of the spinal cord inefficiently and was unable to infect the white matter of the spinal cord. In this article, we report that introducing a Lys at position VP2-141 in virus R5 increases its ability to infect the white matter of the spinal cord. Our results indicate that this amino acid is important for the spread of the virus within the central nervous system.     

Demyelinating and nondemyelinating strains of mouse hepatitis virus differ in their neural cell tropism

Some strains of mouse hepatitis virus (MHV) can induce chronic inflammatory demyelination in mice that mimics certain pathological features of multiple sclerosis. We have examined neural cell tropism of demyelinating and nondemyelinating strains of MHV in order to determine whether central nervous system (CNS) cell tropism plays a role in demyelination. Previous studies demonstrated that recombinant MHV strains, isogenic other than for the spike gene, differ in the extent of neurovirulence and the ability to induce demyelination. Here we demonstrate that these strains also differ in their abilities to infect a particular cell type(s) in the brain. Furthermore, there is a correlation between the differential localization of viral antigen in spinal cord gray matter and that in white matter during acute infection and the ability to induce demyelination later on. Viral antigen from demyelinating strains is detected initially in both gray and white matter, with subsequent localization to white matter of the spinal cord, whereas viral antigen localization of nondemyelinating strains is restricted mainly to gray matter. This observation suggests that the localization of viral antigen to white matter during the acute stage of infection is essential for the induction of chronic demyelination. Overall, these observations suggest that isogenic demyelinating and nondemyelinating strains of MHV, differing in the spike protein expressed, infect neurons and glial cells in different proportions and that differential tropism to a particular CNS cell type may play a significant role in mediating the onset and mechanisms of demyelination.     

Localization of arylsulphatase in neurons

Abstract— Arylsulphatase activity, with 4-methylumbelliferone sulphate as substrate, was measured by a quantitative histochemical method in individual anterior horn nerve cell bodies and adjacent neuropil of man and monkey; and in molecular and granular layers and subjacent white matter of cerebellum of monkey, rat and guinea pig. The activity was much higher in neuronal perikarya than in neuropil, and higher in the granular layer of cerebellum than in the molecular or white matter, thus resembling the distinctive distribution, reported in monkey, of three other lysosomal enzymes, β-galactosidase, β-glucuronidase and α-naphthyl acid phosphatase. One exception was encountered: the white matter of guinea pig cerebellum had more arysulphatase activity than the granular layer. For comparison, other lysosomal enzymes also were measured in rat and guinea pig cerebellum; in these species, α-naphthyl acid phosphatase distribution was found to differ from that of β-galactosidase and arysulphatase, and from the pattern common to four lysosomal enzymes in the monkey.     

Evidence for Ascending and Descending Intraspinal as Well as Primary Sensory Somatostatin Projections in the Rat Spinal Cord

Somatostatin distribution was measured quantitatively in the rat spinal cord by radioimmunoassay. Rostro-caudally, somatostatin content was about 50% higher in lumbar-sacral cord than in cervical or thoracic levels. The dorso-ventral distribution is more uneven: somatostatin is highest in the dorsal horn, where the peptide is 15 times as concentrated as it is in the ventral white matter, the region of lowest concentration. However, measurable amounts of the peptide were found in all regions studied. Dorsal root ganglionectomy decreased somatostatin levels in the dorsal cord, supporting the previously proposed role for this peptide as a primary sensory neurotransmitter or modulator; but somatostatin content also was decreased both rostral and caudal to spinal transection, indicating the presence of ascending and descending somatostatin pathways within the spinal cord. Brain levels did not change. Met-enkephalin and substance P were also measured after the above surgical manipulations. Met-enkephalin content was not altered and substance P content was lowered significantly only after ganglionectomy. Although this study confirms the primary sensory neuron as the origin of a part of spinal cord somatostatin, it further indicates the presence of ascending and descending somatostatin pathways within the rat spinal cord.     

Lamination of spinal cells projecting to the zona incerta of rats

In this study, the lamination patterns of spinal cells projecting to the zona incerta (ZI), intralaminar nuclei and ventral posterior nucleus of the thalamus have been explored. Injections of cholera toxin subunit B or latex beads were made into the ZI, intralaminar and ventral posterior nuclei of Sprague Dawley rats. The brain and spinal cord were then aldehyde fixed and processed using standard methods. Our results show two major findings. First, after injections into the ZI, there is a distinct pattern of lamination of labelled cells in the spinal cord, a pattern that changes across the different levels. At cervical levels, labelled cells are located within the medial region of the deep dorsal horn, while at lumbar and sacral levels, they are found in the intermediate grey matter. These results are similar to those seen after injections into the intralaminar or ventral posterior nuclei, except that in the latter cases, more labelled cells are located in the superficial laminae of the dorsal horn, particularly from the ventral posterior nucleus. Second, the ZI is not associated uniformly with all spinal levels; labelling is heaviest at cervical and lightest at thoracic levels. From each thalamic injection site, labelling is noted on both sides of the spinal cord, with a clear contralateral predominance. In conclusion, the results indicate that the ZI receives a distinct set of spinal projections principally from the cervical level. The particular pattern of lamination of spinal cells projecting to the ZI suggests that the type of information relayed is from deep somatic and/or visceral structures, and probably nociceptive in nature.     

Spread of a neurotropic coronavirus to spinal cord white matter via neurons and astrocytes.

Mouse hepatitis virus strain JHM (MHV-JHM) causes a chronic encephalomyelitis in susceptible mice, with histological evidence of demyelination in the spinal cord. After intranasal inoculation, virus spreads retrogradely to several brain structures along neuroanatomic projections to the main olfactory bulb. In the absence of experimental intervention, mice become moribund before the spinal cord is infected. In this study, infusions of anti-MHV neutralizing monoclonal antibodies were administered to protect mice from the MHV-JHM-induced acute encephalitis and to allow survival until virus spread to the spinal cord. Under these conditions, virus was observed to enter specific layers (primarily laminae V to VII) in the gray matter of the upper spinal cord, consistent with transneuronal spread. While the brain structures which are the sources for virus spread to the spinal cord cannot be determined with certainty, the ventral reticular nucleus is likely to be important since it is consistently and extensively labeled in all mice and receives projections from subsequently infected areas of the spinal cord. After initial entry into the gray matter, virus rapidly spread to the white matter of the spinal cord. During the early stages of this process, extensive infection of astrocytes was noted, suggesting that cell-to-cell spread via these glial cells is an important part of this process. Reports from other laboratories using cultured cells strongly suggested that astrocytes serve as important regulators of oligodendrocyte function and, by extrapolation, have a major role in vivo in the processes of both demyelination and remyelination. Thus, our results not only outline the probable pathway used by MHV-JHM to infect the white matter of the spinal cord but also, with the assumption that infection of astrocytes leads to subsequent dysfunction, raise the possibility that infection of these cells contributes to the demyelinating process.