Flip and Flop Variants of AMPA Receptors in the Rat Lumbar Spinal Cord

The expression of eight messenger RNA splice forms encoding the Flip and Flop variants of AMPA receptor subunits GluR-A to -D in the rat lumbar spinal cord was examined by in situ hybridization using specific oligonucleotides. In the dorsal horn (laminae I, II and III) the predominant mRNA was GluR-B Flip. Much lower levels of GluR-A Flip were found in lamina I and in superficial parts of lamina II outer. In the ventral horn, motor neurons expressed mainly GluR-B Flip, GluR-C Flip and Flop, and GluR-D Flip. Serial sectioning through large motor neurons indicated that a given cell contained, for example, both GluR-C Flip and Flop splice types.     

Expression of NMDA Receptor mRNAs in Rat Motoneurons is Down-regulated after Axotomy

The cytotoxic effects of glutamate via the N -methyl-D-aspartate (NMDA) receptor have been suggested to take part in the events leading to death of motoneurons after neonatal axotomy. By the use of in situ hybridization and immunohistochemistry we have investigated motoneuron mRNA expression of the NMDA receptor subunits NR1, NR2B and NR2D and of the NR1 subunit protein in two lesion models leading to partial motoneuron death: sciatic nerve transection early postnatally in the rat and ventral root avulsion in the adult rat. The results were compared with a lesion model with no subsequent death of motoneurons, i.e. sciatic nerve transection in the adult rat. All lesions were followed by down-regulation of the mRNAs for all studied subunits in severed motoneuron populations; down-regulation was detectable already at early stages postoperatively before any significant death had taken place. The strongest down-regulation was in fact seen in the lesion with the largest loss of motoneurons (ventral root avulsion). The reduction in the expression of NR1 mRNA was paralleled by a decrease in NR1 subunit protein. We conclude that down-regulation of NMDA receptor subunit expression is part of the acute response to axonal injury in motoneurons, whether or not neuronal death follows, and that the susceptibility of lesioned motoneurons to excitotoxic effects should be highest early after axonal injury.     

Regional Spinal Cord and Brain Blood Flows in the Rat

Regional spinal cord blood flow and blood flow in different brain areas were measured in 18 rats by the microsphere technique. By the use of spheres labeled with two different isotopes injected 5 min apart, double determinations were made. The consistency of the results from the two injections was very good. Blood flow was highest in the lumbar part of the spinal cord, somewhat lower in the lower thoracic part, lowest in the upper thoracic part, and again higher in the cervical cord; the mean values of the two determinations were 0.98, 0.74, 0.42, and 0.48 ml · min^-1 · g^-1 tissue for these regions, respectively. The brain areas investigated were the brain stem, cerebellum, and right and left hemispheres; the respective mean values of the two blood flow determinations in these areas were 0.54, 0.58, 0.33, and 0.38 ml · min^-1 · g^-1 tissue.     

Changes in Amino Acid Concentrations over Time and Space around an Impact Injury and Their Diffusion Through the Rat Spinal Cord

Release of amino acids, particularly the neurotoxin glutamate, in and around the site of an experimental spinal cord injury was characterized over time by microdialysis. Increases in amino acid concentrations caused by injury decline steeply and then slowly over distance from the impact area, becoming undetectable beyond about 5 mm from the injury epicenter. Diffusion profiles determined in the cord by administering amino acids through one microdialysis fiber and sampling them in a parallel fiber declined steeply with distance. Distant increases coincided temporally with those in the injury epicenter. We conclude that elevated amino acids more than about 1 mm into the periimpact zone are predominantly released in that region rather than diffusing into it from the trauma epicenter. In the outer areas of lesion development, glutamate does not appear to reach concentrations ordinarily toxic, and elevated concentrations do not persist nearly as long as the therapeutic window of NBQX in any part of the lesion. Therefore, the mechanisms whereby excitatory amino acid antagonists reduce the dimensions of injury lesions are unclear. However, sensitization of neurons following impact injury could be important in amino acid neurotoxicity.     

Local Transcutaneous Cooling of the Spinal Cord in the Rat: Effects on Long-Term Outcomes After Compression Spinal Cord Injury

This study tested efficiency of a novel thermoelectric cooler for local transcutaneous spinal cord cooling. Spinal cord compression was made by epidural balloon inflation at T8-T9 level of the spinal cord. Experimental animals (n = 20) were divided into two groups. In the hypothermic group, local cooling started 25 min after spinal cord injury and lasted for 1 h with paravertebral temperature maintained at 28.5°C (±0.3). Normothermic group underwent identical procedures, but their temperature was maintained normothermic. The assessment of neurologic recovery was performed once a week during a 4 weeks survival period. After 4 weeks animals were sacrificed and the extent of the spinal cord lesion morphometrically evaluated. There were no statistically significant intergroup differences in BBB scores and preserved volumes of the spinal cord tissue. In consecutive cross-sectional areas, hypothermic animals had significantly more preserved white matter at the cranial periphery of the lesion. It was concluded that mild posttraumatic hypothermia (31.8°C) had some protective effect on tissue loss after spinal cord injury but this effect was not associated with functional improvement.     

Biochemical and pharmacological evidence of a functional role of AMPA receptors in motor neuron dysfunction in mnd mice

We studied ionotropic glutamate receptor subtypes and the effect of chronic treatment with NBQX [6-nitro-7-sulphamoyl-benzo(F)quinoxaline-2,3-dione], a selective (rs)-alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor antagonist, in the spinal cord of mnd mice. NBQX (8 mg/kg daily i.p. for 3 weeks starting from 24 weeks old) significantly improved the behavioural scores (hind leg extension reflex, cage rung grasping and gait) in mnd mice, measured after the last drug injection, and increased the number of mice with 'normal' gait (from 50% to 90%, P < 0.05). Receptor binding autoradiography of the competitive N-methyl-D-aspartate (NMDA) antagonist, [3H]CGP 39653, of [3H]AMPA and [3H]kainic acid in spinal cord sections, measured after 1 week of drug washout, were not significantly different in control and mnd mice, and were not modified by NBQX. GluR2/3 immunoreactivity, assessed using Western blotting, was significantly enhanced (by 59%, P < 0.01) in the spinal cord but not in the brain of 28-week-old mnd mice compared to age-matched control mice. NBQX treatment increased GluR2/3 immunoreactivity in the spinal cord of control mice and mnd mice by 327 +/- 74% (P < 0.01) and 212 +/- 52% (P < 0.01), respectively. The changes in GluR2/3 subunits may involve adaptive mechanisms of the receptor and play some role in the protective effect of NBQX. These findings suggest that selective antagonism of ionotropic non-NMDA receptors may be of value in the treatment of motor neuron disease.     

Neuronal Apoptosis and Necrosis Following Spinal Cord Ischemia in the Rat

We examined the characteristics of neuronal death induced by ischemia in the spinal cord. Spinal cord ischemia was induced in Long–Evans rats by occlusion of the descending aorta with a 2F Fogarty catheter for 20 min (model 1) or more limited aortic occlusion (15 min) coupled with blood volume reduction (model 2); rats were sacrificed 6 h–7 days later. The animals developed variable paraparesis in model 1 and reliable paraplegia in model 2. The extent of histopathological spinal cord damage, being maximal in the lumbar cord, correlated well with the severity of paraparesis. Two distinct types of spinal cord neuronal death were observed, consistent with necrosis and apoptosis. Neuronal necrosis was seen in gray matter laminae 3–7, characterized by the rapid (6 h) onset of eosinophilia on hematoxylin/eosin-stained sections, and gradual (1–7 days) development of eosinophilic ghosting. Although TUNEL positivity was present, disintegration of membranes and cytoplasmic organelles was seen under electron microscopy. Neuronal apoptosis was seen after 1–2 days in dorsal horn laminae 1–3, characterized by both TUNEL positivity and electron microscopic appearance of nuclear chromatin aggregation and the formation of apoptotic bodies. DNA extracted from the ischemic lumbar cord showed internucleosomal fragmentation (laddering) on gel electrophoresis. These data suggest that distinct spinal cord neuronal populations may undergo necrosis and apoptosis following transient ischemic insults.     

Immunohistochemical Localization of Kininogen in Rat Spinal Cord and Brain

Kininogen localization has been determined by immunocytochemistry in rat spinal cord and brain using a kinin-directed kininogen monoclonal antibody. In the spinal cord, there were immunostained neurons and fibers in laminae I, II, VII, and IX, intensely stained fibers in the superficial layers of the dorsal horn, and immunoreactive glial and endothelial cells. Small neurons, satellite cells, and Schwann cells immunostained distinctly in the dorsal root ganglion. In the brain stem, there were immunoreactive neurons and fibers in the tractus solitarius and nucleus, trigeminal spinal tract and nuclei, periaqueductal gray matter, vestibular nuclei, cochlear nuclei, trapezoid body, medial geniculate nucleus, and red nucleus. Immunostained neurons and fibers were also found in cerebellum (dentate nucleus), cerebral cortex (layers III and V), hippocampus (pyramidal cell layer), and corpus callosum. Glia and endothelial cells stained in all brain regions. The widespread location of kininogen in neurons and their processes, as well as in glial and endothelial cells, indicates more than one functional role, including those proposed as a mediator, a calpain inhibitor, and a kinin precursor, in a variety of neural activities and responses.     

Membrane Potential Oscillations of Neonatal Rat Spinal Motoneurons Evoked by Electrical Stimulation of Dorsal Root Fibres

Intracellular recording from lumbar motoneurons of the neonatal rat isolated spinal cord bathed in standard saline solution was used to study membrane potential oscillations which accompanied the decay phase of excitatory postsynaptic potentials (EPSP) induced by single electrical pulses to an adjacent dorsal root. About 60% of motoneurons displayed rhythmic oscillations of 10 ± 2 mV maximal amplitude and 7 ± 0.5 Hz frequency. Ability to generate oscillations could not be correlated to the cell membrane properties or to the age of the preparation (5–13 days). The oscillation frequency was independent of membrane potential (-100 to -45 mV) or of the intensity of dorsal root stimuli. The oscillation amplitude was linearly related to the cell potential within the same voltage level. Fast Fourier transform analysis showed that the power spectrum of oscillations peaked at ˜8 Hz. Electrically evoked activities and spontaneous events displayed similar cut-off frequencies. When the cell membrane potential was steadily depolarized, a hyperpolarizing pulse applied during the decay phase of the EPSP promptly revealed the presence of oscillatory behaviour. Pharmacological block of neurokinin-1 or N -methyl-D-aspartate receptors depressed the decay phase of the EPSP and the associated oscillatory responses. It is suggested that rhythmic oscillations were probably due to summated synaptic potentials generated at the premotoneuron level and are perhaps of functional relevance to motoneuron behaviour.     

大鼠脊髓冲击伤模型制作方法的改良探索

目的:探索一种制作稳定性强、重复性好的脊髓损伤模型的简易方法。方法:应用简易重物坠落打击装置建立大鼠脊髓中度损伤组(5g×8cm)、重度损伤组(5g×16cm)模型和假手术组,每组大鼠均n=10。通过运动功能评分法(BBB)评分、苏木精-伊红染色观察脊髓损伤后两组大鼠功能及病理的变化特点,以评价该制作方法的可靠性。结果:BBB评分显示两组大鼠后肢运动功能均有不同程度的恢复,但中度损伤大鼠的恢复明显优于重度损伤大鼠(P<0.05)。组织病理学观察显示脊髓损伤后脊髓结构紊乱,有胶质瘢痕及空洞形成,重度损伤组脊髓空洞面积明显大于中度组(P<0.05)。结论:该方法制作的大鼠脊髓模型能将不同打击力度造成的损伤区分开,并且模型的行为学与病理学结果相吻合,说明此模型具有良好的稳定性、重复性和一致性,适合脊髓损伤实验研究的应用。     

Prostaglandin E2 Receptors in the Chicken Spinal Cord: 2. Radioautographic Localization

High and low affinity binding sites for PGE2 were localized at the cellular level by means of radioautography. Two types of radioautographic reactions were observed in cryostat sections incubated with 0.6 nM [3H]PGE2: (i) a diffuse reaction scattered over the neuropil; (ii) a stronger reaction over the perikarya of some motoneurons. However, not all the motoneurons were equally labelled; the density of silver grains increased 12 times from poorly labelled to highly labelled cells. After incubation with an excess of unlabelled PGE2 the labelling was drastically reduced over motoneurons and the neuropil, so that the specific binding corresponded to 90 and 85% respectively of the total binding. Two facts indicated that the motoneurons possessed mainly high affinity binding sites which could be saturated by PGE2 concentrations in the nanomolar range: (i) a five-fold increase of [3H]PGE2 concentration (from 0.6 to 3 nM) produced only a 1.5 times increase in the density of silver grains over the perikarya; (ii) incubation in presence of 14 nM unlabelled PGE2 induced a drastic reduction of 0.6 nM [3H]PGE2 binding to the motoneurons. The presence of high affinity binding sites in motoneurons suggests that PGE2 could act as a modulator of spinal reflexes.     

Prostaglandin E2 Receptors in the Chicken Spinal Cord: 1. Biochemical Characterization

Prostaglandins (PGs) are neuroactive substances which act in the vicinity of their site of synthesis through receptors coupled to G-proteins. Since large amounts of PGE2 can be synthesized by chicken spinal cord, binding sites for PGE2 were looked for in various cell fractions of spinal cord. In the 17 000 g pellet incubated with 0.3 nM [3H]PGE2, 70% of ligand was specifically bound. Two types of PGE2 binding site were characterized (i) high affinity, low capacity binding sites (KD1 1.34 nM, Bmax1 34.5 fmol/mg prot); (ii) low affinity, high capacity binding sites (KD2 2.23 microM, Bmax2 13.2 pmol/mg prot). The high affinity binding sites fulfil several requirements for being receptors to PGE2: (i) since the KD1 is increased in the presence of the GTP analogue, Gpp(NH)p, these binding sites would be regulated by a G-protein; (ii) a desensitization was obtained by an excess of unlabelled PGE2 and reversed by Gpp(NH)p; (iii) the competition experiments between PGE2 and various prostanoids pointed to PGE2 receptors such as EP2 or EP3. The receptor characteristics of the low-affinity binding sites were not investigated. Hence, our results support the presence of two types of PGE2 binding site in the chicken spinal cord; a high affinity site, which corresponds to a PGE2 receptor responding to nanomolar concentrations and a low affinity site sensitive to micromolar concentrations of PGE2.     

An NK1 Receptor-dependent Component of the Slow Excitation Recorded Intracellularly from Rat Motoneurons Following Dorsal Root Stimulation

Intracellular recording from lumbar motoneurons of the neonatal rat spinal cord in vitro was used to study how recently developed non-peptide antagonists such as SR-140333 and SR-48698, known to block distinct subtypes of tachykinin receptors peripherally, might affect synaptic transmission elicited by electrical stimulation of dorsal root fibres. SR-140333 (1 μM) preferentially antagonized responses mediated by an exogenously applied agonist acting on the NK₁ receptor subclass, while SR-48968 (0.5 μM) preferentially reduced responses mediated by an exogenously applied agonist acting on the NK₂ receptor subclass. SR-48968 did not affect fast or slow excitatory postsynaptic potentials (EPSPs) or ‘wind-up’responses induced by repetitive, low-frequency stimulation (mimicking certain types of nociceptive input); binding studies using this radiolabelled ligand disclosed specific binding activity (21 fmol/mg protein) selectively displaced by an NK₂ receptor agonist. SR-140333 reduced the late component of fast and slow EPSPs, and of wind-up. Pharmacological block of ionotropic glutamate receptors abolished all dorsal root-evoked EPSPs. In comparison to glutamate receptor blockers, SR-140333 was a weaker antagonist of slow synaptic responses, though it displayed preferential antagonism towards some components of the wind-up phenomenon. The present results provide evidence obtained with a novel NK₁ antagonist that a neuropeptide (presumably substance P), although not directly released by primary afferents onto motoneurons, is a neurotransmitter (acting via NK₁ receptors) in the pathway mediating slow synaptic responses of motoneurons, and is presumably involved in signalling nociceptive inputs from the periphery.     

AMPA Receptors are Coupled to the Nitric Oxide/Cyclic GMP Pathway in Cerebellar Astroglial Cells

In cultured rat cerebellar astroglia kainate induces cGMP formation with low potency (EC₅₀ 310 μM). In the presence of cyclothiazide, a blocker of α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptor desensitization, the effect of kainate was potentiated and glutamate and AMPA elicited large increases (>100-fold) in cGMP levels. The response to all three agonists was abolished by the nitric oxide synthase inhibitor N^G-nitro-L-arginine and required extracellular calcium. Uptake of Co²⁺ was induced by AMPA in a limited population of astroglial cells and this effect was potentiated by cyclothiazide. These results indicate that calcium-permeable AMPA receptors mediate stimulation of nitric oxide formation in cerebellar astroglia. This effect may be relevant for glutamate-dependent synaptic plasticity processes in the cerebellum.