Sections Of Rat Spinal Cord Research Articles

Detection of Neuronal and Glial Antigens After Decalcification in Formic Acid Solution and Fixation in Zinc-Ethanol-Formaldehyde

The aim of the present work was to identify the possibility of immunocytochemical detection of various tissue antigens on paraffin sections of rat spinal cord surrounded by spinal bony tissue and adjacent muscles fixed in zinc-ethanol-formaldehyde after decalcification with formic acid in adult (n = 5) and neonatal (n = 2) Wistar rats. These studies used antibodies to glial fibrillary acidic protein, neurofilaments, neuronal nuclear protein, tyrosine hydroxylase, the synaptic vesicle protein synaptophysin, and the intermediate filament protein vimentin. The combination of fixation in zinc-ethanol-formaldehyde and decalcification in 21% formic acid solution provided optimal preservation of the antigens of interest.

Transplantation and follow-up of human iPSc-derived neural progenitors in a rat model of ALS

Transplantation and follow-up of human iPSc-derived neural progenitors in a rat model of ALS

Expression and ultrastructural localization of Mint2 in the spinal cord of rats

Mint protein family, as adaptor molecules, contains three members, Mint1, Mint2 and Mint3. Although Mint3 is ubiquitously expressed, Mint1 and Mint2 have been reported to express specifically in neuron. Here we demonstrated Mint1 and Mint2 expression pattern in rat spinal cord. The protein level of Mint2 was found to be higher than that of Mint1 in rat spinal by western blot. In an attempt to know Mint2 distribution in the spinal cord of rat, in situ hybridization was carried out, Mint2 mRNA was showed to be ubiquitously distributed in cervical, thoracic and lumbar sections of rat spinal cord, and high intensive signal was detected in motor neurons. These were further confirmed by fluorescent immunohistochemistry, Mint2 was also found to exist throughout gray matter especially motor neurons where Mint2 was mainly located in perikaryon, however, Mint1 was showed to be relatively lower. By electron microscope, Mint2 was found to be mainly located in vesicles in perikaryon in motor neuron of lumbar section, and at the same time Mint2 was located in axons in myelin and presynaptic terminals. These data suggest that Mint2 may play more important role in spinal cord than the other two family members.

SIMS and MALDI MS imaging of the spinal cord

The application of MS to imaging, or MS imaging (MSI), allows for the direct investigation of tissue sections to identify biological compounds and determine their spatial distribution. We present an approach to MSI that combines secondary ion MS (SIMS) and MALDI MS for the imaging and analysis of rat spinal cord sections, thereby enhancing the chemical coverage obtained from an MSI experiment. The spinal cord is organized into discrete, anatomically defined areas that include motor and sensory networks composed of chemically diverse cells. The MSI data presented here reveal the spatial distribution of multiple phospholipids, proteins, and neuropeptides obtained within single, 20 mum sections of rat spinal cord. Analyte identities are initially determined by primary mass match and confirmed in follow-up experiments using LC MS/MS from extracts of adjacent spinal cord sections. Additionally, a regional analysis of differentially localized signals serves to rapidly screen compounds of varying intensities across multiple spinal regions. These MSI analyses reveal new insights into the chemical architecture of the spinal cord and set the stage for future imaging studies of the chemical changes induced by pain, anesthesia, and drug tolerance.

Select ionotropic glutamate AMPA/kainate receptors are expressed at the astrocyte–vessel interface

Ionotropic AMPA/kainate (KA) glutamate receptors are important for vasculature–astrocyte interaction in the central nervous system (CNS). To date, little is known about the expression of AMPA/KA receptors in the perivascular environment. Using double labeling immunohistochemistry on mouse and rat spinal cord sections, we show first evidence that perivascular astrocytic processes express specifically GluR4 (AMPA) and GluR5 (KA) subunits in both species. In contrast, astrocytes display GluR2/3 and GluR6/7 immunoreactivity throughout the cell but not limited to vascular profiles. Interestingly, KA2 immunoreactivity appears perivascular only in rats but not in mice. No perivascular labeling for any of the probed subunits was found in peripheral sciatic nerve. These data suggest that, in both mice and rats, few and select AMPA/KA subunits are uniquely positioned to mediate the effects of glutamate on astrocyte–vessel interactions.

Role of adenosine receptors in spinal G-protein activation after peripheral nerve injury.

Spinally injected adenosine induces antinociception in animal models of neuropathic but not acute pain. The reasons for this discrepancy remain unclear. Adenosine receptors are coupled to G proteins, and increased efficiency of adenosine-induced G-protein activity in neuropathic pain could contribute to the antinociceptive effect of adenosine. In this study the authors used [(35)S]guanosine-5'-O-(3-thiotriphosphate) ([(35)S]GTP gamma S) autoradiography in rat spinal cord sections to test this possibility. The spinal cords of normal animals and those that underwent left L5 and L6 spinal nerve ligation (SNL) were removed and immediately frozen. Horizontal spinal cord sections were cut and mounted on chrom-alum gelatin-subbed slides. Sections were incubated with guanosine diphosphate, [(35)S]GTP gamma S, the adenosine A1 agonist R-N6-phenylisopropyladenosine, and various other drugs, apposed to films, and analyzed. Baseline and R-N6-phenylisopropyladenosine-stimulated [(35)S]GTP gamma S binding was predominantly localized to the superficial dorsal horns of both normal and SNL animals. This binding was significantly increased in SNL compared with normal animals. In contrast, no difference in R-N6-phenylisopropyladenosine-stimulated [(35)S]GTP gamma S binding was observed between SNL and normal animals. Blockade of adenosine A1 receptors by 1,3-dipropyl-8-cyclopentylxanthine, or adenosine destruction by added adenosine deaminase, reduced the increased basal activity in SNL to baseline levels of normal dorsal horns, whereas atropine and naloxone had no effect. This study shows an increased basal G-protein activity in lumbar spinal cords during conditions of SNL. The data suggest that increased adenosine release during conditions of SNL results in an increased basal activity of G proteins in the spinal cord during neuropathic pain.

Anticonvulsant activity of a nonpeptide galanin receptor agonist

Galanin is a neuropeptide with a wide variety of biological functions, including that of a strong endogenous anticonvulsant. No nonpeptide ligands, capable of activating galanin receptors, are available today. Based on known pharmacophores of galanin, a combinatorial library was designed, synthesized, and screened at the rat hippocampal galanin receptor. A low molecular weight galanin receptor agonist, 7-((9-fluorenylmethoxycarbonyl)cyclohexylalanyllysyl)amino-4-methylcoumarin (galnon) was found to displace (125)I-galanin with micromolar affinity at Bowes cellular and rat hippocampal membranes. Autoradiographic binding assay on rat spinal cord sections confirmed the ability of galnon to displace (125)I-galanin from its binding sites. Galnon inhibited adenylate cyclase activity, suggesting an agonist action at galanin receptors. When injected i.p. galnon reduced the severity and increased the latency of pentylenetetrazole-induced seizures in mice and reversed the proconvulsant effects of the galanin receptor antagonist M35, injected into a lateral ventricle. Intrahippocampal injection of galnon also shortened the duration of self-sustaining status epilepticus in rats, confirming its agonist properties in vivo. Pretreatment of rats with antisense peptide nucleic acid targeted to galanin receptor type 1 mRNA abolished the effect of galnon, suggesting mediation of its anticonvulsant properties through this receptor subtype. These findings introduce a systemically active nonpeptide galanin agonist anticonvulsant.

Coexistence of kappa- and delta-opioid receptors in rat spinal cord axons

It has been found that heterodimers of kappa- and delta-opioid receptors can occur in vitro, but it has been unclear whether they also occur in intact animals. In the present study we examined whether kappa-delta heterodimers might occur in vivo by staining for these receptors with two-color fluorescence immunocytochemistry. Sections of rat spinal cord were double-stained using rabbit anti-kappa opioid receptor combined with rat anti-delta-opioid receptor. It was found that axons in the superficial dorsal horn of the spinal cord were double-labeled. In addition, structures within axonal varicosities were sometimes double-labeled. We conclude that single axons, and single structures within axons, express both kappa- and delta-opioid receptors. These observations are consistent with heterodimers of these receptors existing in vivo.

Nociceptin receptors in the rat spinal cord during morphine tolerance

The anatomical localization of nociceptin receptors was examined by in vitro quantitative autoradiography techniques in rat spinal cord sections by using [ 125 I –Tyr 14]nociceptin. [ 125 I –Tyr 14]nociceptin appeared to interact with a single class of binding sites ( K D=0.1 nM) present in the grey matter in all laminae of the spinal cord from cervical to sacral levels. Pre-incubation of sections in the presence of 150 mM NaCl, did not modify the radioligand affinity but significantly augmented the number of accessible binding sites and increased specific binding of [ 125 I –Tyr 14]nociceptin differentially on each laminae. In particular, the superficial layers of the dorsal horn exhibited the highest density of sites after pre-wash. Continuous intrathecal infusion of morphine produced a tolerance accompanied by a significant increase in nociceptin site density in the superficial layers. Thus, nociceptin binding sites may have different properties dependent upon the layer and may be up-regulated during the process of opioid-induced tolerance.

Increased calpain expression in activated glial and inflammatory cells in experimental allergic encephalomyelitis.

In demyelinating diseases such as multiple sclerosis (MS), myelin membrane structure is destabilized as myelin proteins are lost. Calcium-activated neutral proteinase (calpain) is believed to participate in myelin protein degradation because known calpain substrates [myelin basic protein (MBP); myelin-associated glycoprotein] are degraded in this disease. In exploring the role of calpain in demyelinating diseases, we examined calpain expression in Lewis rats with acute experimental allergic encephalomyelitis (EAE), an animal model for MS. Using double-immunofluorescence labeling to identify cells expressing calpain, we labeled rat spinal cord sections for calpain with a polyclonal millicalpain antibody and with mAbs for glial (GFAP, OX42, GalC) and inflammatory (CD2, ED2, interferon gamma) cell-specific markers. Calpain expression was increased in activated microglia (OX42) and infiltrating macrophages (ED2) compared with controls. Oligodendrocytes (galactocerebroside) and astrocytes (GFAP) had constitutive calpain expression in normal spinal cords whereas reactive astrocytes in spinal cords from animals with EAE exhibited markedly increased calpain levels compared with astrocytes in adjuvant controls. Oligodendrocytes in spinal cords from rats with EAE expressed increased calpain levels in some areas, but overall the increases in calpain expression were small. Most T cells in grade 4 EAE expressed low levels of calpain, but interferon gamma-positive cells demonstrated markedly increased calpain expression. These findings suggest that increased levels of calpain in activated glial and inflammatory cells in EAE may contribute to myelin destruction in demyelinating diseases such as MS.

Neuropeptide FF receptors in rat brain: a quantitative light-microscopic autoradiographic study using [125I][D.Tyr1, (NMe)Phe3]NPFF.

The anatomical localization of neuropeptide FF receptors was examined by in vitro autoradiography techniques in rat brain sections by using [125I][D.Tyr1, (NMe)Phe3]NPFF. The specific binding of [125I][D.Tyr1, (NMe)Phe3]NPFF reached 90% of the total binding at 0.05 nM in rat spinal cord sections. Up to 40% of the specific binding of[125I][D.Tyr1, (NMe)Phe3]NPFF to rat spinal cord sections was still detectable following fixation with glutaraldehyde. Afterwards, the distribution of NPFF receptors was studied by light microscopy and their densities by microdensitometry with an image analysis system. In the light microscope, [125I][D.Tyr1, (NMe)Phe3]NPFF labelling appeared more or less uniformly distributed over nerve-cell bodies and surrounding neuropil. High concentrations of binding sites were detected in the presubiculum, parafascicular thalamic nucleus, gracile nucleus, spinal trigeminal tract nucleus, and a number of brainstem nuclei, with virtually no labelling in the cerebellum. In several areas a rostrocaudal gradient of sites concentration was observed. Neuropeptide FF receptors are well-placed to control incoming sensory and autonomic information processing. In contrast, the more recently developed areas of the forebrain possessed low density of sites. The distribution of [125I][D.Tyr1, (NMe)Phe3]NPFF binding sites should suggest anatomical substrates for the actions of neuropeptide FF.

Relationship between glial organization and the establishment of nerve tracts in rat spinal cord.

Embryo and adult rat spinal cord sections immunostained for S-100 protein, vimentin, glial fibrillary acidic protein (GFAP), or phosphorylated 160 kDa neurofilament protein (NF), were used to study glial participation in nerve tissue assembly. Radial glial fibers (RGF) expressed vimentin since E12. In the peripheral ventral zone, RGF formed cephalocaudal plates (CP) within the white matter, ensheathing developing axonal tracts first expressing NF at E13. As axonal tracts increased, CP became denser and extended ventrolaterally. S-100 protein appeared at E17 in the midline and, at E18, it overlapped vimentin expression. Astrocytes expressed GFAP since E20. We conclude that the pattern found in the spinal cord results from interactions between developing axonal tracts and radial glia, and that it could set the structural basis for the organized assembly of the spinal cord found in adults.

Choline acetyltransferase and NADPH diaphorase are co-expressed in rat spinal cord neurons

Phenotypic diversity underlies the complex functioning of the nervous system. One characteristic in which neurons differ from one another is the kind of molecules that they use for intercellular signalling. The classical neurotransmitter acetylcholine, synthesized by the enzyme choline acetyltransferase, is used by five groups of neurons in the rat spinal cord. Another messenger is nitric oxide, which is synthesized by nitric oxide synthase. Neurons that express nitric oxide synthase can be stained specifically by NADPH diaphorase histochemistry. In the spinal cord, approximately five groups of neurons are labeled by the diaphorase reaction, and some of these populations overlap with cholinergic groups. To determine the proportions of neurons that co-express choline acetyltransferase and nitric oxide synthase, we performed choline acetyltransferase immunocytochemistry and diaphorase histochemistry on single sections of rat spinal cord. Some cell types were single-labeled: somatic motor neurons were choline acetyltransferase-immunoreactive only, and neurons in lamina II were diaphorase-positive only. Four cell groups included double-labeled cells. Autonomic motor neurons were either double-labeled (62%) or choline acetyltransferase-only (37%), partition cells in lamina VII were double-labeled (54%) or choline acetyltransferase-only (45%), neurons in laminae III–V of the dorsal horn were double-labeled (70%) or diaphorase-only (27%), and neurons surrounding the central canal were double-labeled (56%), choline acetyltransferase-only (23%) or diaphorase-only (21%). These data indicate that certain spinal cord populations may be heterogeneous with regard to the intercellular messenger phenotypes involving acetylcholine and nitric oxide. Because phenotypic diversity of messenger systems contributes to physiological differences, our findings raise the possibility that some cells perform different functions than other cells within the same general group. In addition, the results also suggest a possible basis for the differential vulnerability of certain types of neurons in degenerative neurological disorders, such as amyotrophic lateral sclerosis.

Light and electron microscopic immunocytochemical localization of AMPA‐selective glutamate receptors in the rat spinal cord

alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)-type glutamate receptors are probably the most widespread excitatory neurotransmitter receptors of the central nervous system, and they play a role in most normal and pathological neural activities. However, previous detailed studies of AMPA subunit distribution have been limited mainly to the brain. Thus, a comprehensive study of AMPA receptor subunit distribution was carried out on sections of rat spinal cord and dorsal root ganglia, which were immunolabeled with antibodies made against peptides corresponding to C-terminal portions of the AMPA receptor subunits: GluR1, GluR2/3, and GluR4. In the spinal cord, labeling was most prominent in the superficial dorsal horn, motoneurons, and nuclei containing preganglionic autonomic neurons. Immunostaining also was observed in neurons in other regions including those known to contain Renshaw cells and Ia inhibitory cells. Although overall immunostaining was lighter with antibody to GluR1 than with GluR2/3 and 4, there were neurons that preferentially stained with antibody to GluR1. These "GluR1 intense" neurons were usually fusiform and most concentrated in lamina X. In dorsal root ganglia, immunostaining of ganglion cell bodies was moderate to dense with antibody to GluR2/3 and light to moderate with antibody to GluR4. Possible neuroglia in the spinal cord (mainly GluR2/3 and 4) and satellite cells in dorsal root ganglia (GluR4) were immunostained. Electron microscopic studies of the superficial dorsal horn and lateral motor column showed staining that was restricted mainly to postsynaptic densities and associated dendritic and cell body cytoplasm. In dorsal horn, colocalization of dense-cored vesicles with clear, round synaptic vesicles was observed in unstained presynaptic terminals apposed to stained postsynaptic densities. Subsynaptic dense bodies (Taxi-bodies) were associated with some stained postsynaptic densities in both the superficial dorsal horn and lateral motor column. Based on several morphological features including vesicle structure and presence of Taxi-bodies, it is likely that at least some of the postsynaptic staining seen in this study is apposed to glutamatergic input from primary sensory afferent terminals.

Localisation of glutamate receptor binding sites and mRNAS to the dorsal horn of the rat spinal cord

The autoradiographic distribution of binding sites for [ 3H]AMPA, [ 3H]kainate, [ 3H]MK-801 and [ 3H] L-glutamate and the in situ hybridization of GluR-A, GluR-B, GluR-C and GluR-D in rat spinal cord sections were determined. All of the radioligands had similar localisations with the dorsal horn being the most intensely labelled region. In the in situ hybridization experiments only the GluR-B mRNA was detected at high levels in the spinal cord with predominant expression in the substantia gelatinosa. These results support electrophysiological evidence for the presence of glutamate activated neurones in the spinal cord and suggest that these excitatory pathways are confined mainly to the dorsal horn.

The extracellular matrix of rat spinal cord: A comparative study on the localization of hyaluronic acid, glial hyaluronate-binding protein, and chondroitin sulfate proteoglycan

The localization of hyaluronic acid (HA), glial hyaluronate-binding protein (GHAP), and chondroitin sulfate (CS) proteoglycan was compared in cryostat sections of rat spinal cord. HA, GHAP, and CS proteoglycan were similarly distributed in white matter where they surrounded myelinated axons. In gray matter, large motoneurons were surrounded by a rim of reaction product in sections stained for HA and CS proteoglycan. GHAP immunoreactivity as well as HA had disappeared in hyaluronidase-digested sections, while CS proteoglycan immunoreactivity was not abolished under these conditions.

Immunocytochemical localization of neuromedin K (neurokinin B) in rat spinal ganglia and cord

Specific antisera directed against substance P and neuromedin K (neurokinin B) have been used in double-label immunofluorescence studies to unambiguously localize these two neuropeptides of the tachykinin family in single tissue sections of rat spinal cord and dorsal root ganglia. Substance P-like immunoreactivity (SPLI) is present but neuromedin K-like immunoreactivity (NMKLI) is undetectable in dorsal root ganglia. Both peptides are present in the spinal cord, but NMKLI is largely restricted to the dorsal gray while SPLI shows a broader distribution. In the spinal gray, NMKLI coexists with SPLI in some, but not all, fibers. While substance P in the dorsal spinal cord is largely of primary afferent origin, neuromedin K appears to originate largely from intrinsic spinal neurons.

Changes in permissivity for neuronal attachment and neurite outgrowth of spinal cord grey and white matters during development: a study with the ‘cryoculture’ bioassay

We have used the recently developed cryoculture bioassay (Carbonetto et al., J. Neurosci., 7 (1987) 610–620) to document changes during development of CNS tissular ability to support nerve fiber growth. Neuronal attachment and neurite outgrowth of purified neurons cultured on tissue sections of rat spinal cord at various stages of development were quantified. Nerve fiber growth permissivity increased during embryonic stages, reaching at postnatal days 2–4 (P2–P4) a maximum value, higher than that found on adult PNS tissue sections. This permissivity diminished rapidly thereafter, indicating that early postnatally, the nerve fiber growth supporting ability of the CNS environment shifts abruptly from an increasingly permissive mode to an increasingly non-permissive status. Furthermore, after P4, neurite outgrowth permissivity diminished in parallel on white and grey matters, whereas neuronal attachment declined much more drastically on white matter than on grey matter. This indicates that the progressive loss of spinal cord ability to support nerve fiber growth is attributable to both grey and white matters. In several instances it also appeared that neuronal adhesion was not necessarily followed by a comparable level of nerve fiber growth, suggesting that these two processes could be regulated by different factors.

Axons modulate myelin protein messenger RNA levels during central nervous system myelination in vivo

Expression of myelin protein genes by myelinating Schwann cells in vivo is dependent on axonal influences. This report investigated the effect of axons on myelin protein mRNA levels in the central nervous system (CNS). In situ hybridization studies of rat spinal cord sections localized mRNAs encoding proteolipid protein (PLP) and myelin basic protein (MBP) 20 and 40 days after unilateral rhizotomy. Compared with control tissue, hybridization intensity was reduced in transected tissue, but there was little change in the number of oligodendrocytes labeled. Cellular RNA was extracted from transected and age-matched control optic nerves 5, 10, 20, and 40 days after surgery, and levels of the following mRNAs were determined by slot blot procedures: PLP, MBP, myelin-associated glycoprotein (MAG), and 2',3' cyclic nucleotide 3'-phosphodiesterase (CNP). In transected nerves, PLP and MBP mRNA levels were approximately 85%, 45%, and 25% of control values at 5, 20 and 40 days posttransection, respectively. Axonal transection had a lesser effect on CNP and MAG mRNA levels, which declined to approximately 60% of control levels at 40 days. Immunocytochemical studies indicated that the number of oligodendrocytes was not decreased 40 days after optic nerve transection. These data demonstrate that axons modulate myelin protein mRNA levels in oligodendrocytes. In contrast to Schwann cells, however, oligodendrocytes continue to express significant levels of myelin protein mRNA in vivo following loss of axonal contact.

Phencyclidine and sigma receptors in rat spinal cord: Binding characterization and quantitative autoradiography

These experiments were designed to compare phencyclidine (PCP) and sigma (sigma) receptor binding sites in the rat spinal cord by using receptor binding and autoradiographic techniques. Binding sites for 3H-TCP (3H-1-[1-(2-thienyl)cyclohexy]piperidine), a PCP receptor agonist, and (+)3H-3-PPP (3H-(+)-3-(3-hydroxyphenyl)-N-(1-propyl)piperidine), a sigma receptor agonist, in the rat spinal cord were shown to represent two populations of recognition sites. Inhibition studies revealed that ligands with high affinity for the PCP receptor (MK-801 and PCP) were potent competitors at 3H-TCP binding sites whereas the putative sigma receptor ligands (+/-)pentazocine and haloperidol were potent competitors at (+)3H-3-PPP binding sites. The autoradiographic distribution of 3H-TCP and (+)3H-3-PPP binding sites in adjacent sections of rat spinal cord demonstrated the occurrence of two distinct populations of binding sites. 3H-TCP binding sites were localized primarily in laminae I and II in cervical and thoracic spinal segments. Binding sites in lamina I decreased in density along a rostral to caudal gradient in the spinal cord. The highest density of (+)3H-3-PPP binding sites was found in the ventral horn (lamina VIII and IX) and over perikarya in dorsal root ganglia. Significantly elevated densities of (+)3H-3-PPP binding sites were also found in lamina X within thoracic and lumbar segments and in the intermediolateral cell column. The results of the present study show that PCP and sigma receptor binding sites are differentially localized in the rat spinal cord and suggest that separate binding sites exist for PCP and sigma agonists.