Cord Slices Research Articles

Rebound depolarization of substantia gelatinosa neurons and its modulatory mechanisms in rat spinal dorsal horn

To investigate the rebound depolarization of substantia gelatinosa (SG) neurons in rat spinal dorsal horn and explore its modulatory mechanisms to provide better insights into rebound depolarization-related diseases. Parasagittal slices of the spinal cord were prepared from 3- to 5-week-old Sprague-Dawley rats. The electrophysiologic characteristics and responses to hyperpolarization stimulation were recorded using whole-cell patch-clamp technique. The effects of hyperpolarization-activated cyclic nucleotide gated cation (HCN) channel blockers and T-type calcium channel blockers on rebound depolarization of the neurons were studied. A total of 63 SG neurons were recorded. Among them, 23 neurons showed no rebound depolarization, 19 neurons showed rebound depolarization without spikes, and 21 neurons showed rebound depolarization with spikes. The action potential thresholds of the neurons without rebound depolarization were significantly higher than those of the neurons with rebound depolarization and spikes (-28.7∓1.6 mV vs -36.0∓2.0 mV, P<0.05). The two HCN channel blockers CsCl and ZD7288 significantly delayed the latency of rebound depolarization with spike from 45.9∓11.6 ms to 121.6∓51.3 ms (P<0.05) and from 36.2∓10.3 ms to 73.6∓13.6 ms (P<0.05), respectively. ZD7288 also significantly prolonged the latency of rebound depolarization without spike from 71.9∓35.1 ms to 267.0∓68.8 ms (P<0.05). The T-type calcium channel blockers NiCl2 and mibefradil strongly decreased the amplitude of rebound depolarization with spike from 19.9∓6.3 mV to 9.5∓4.5 mV (P<0.05) and from 26.1∓9.4 mV to 15.5∓5.0 mV (P<0.05), respectively. Mibefradil also significantly decreased the amplitude of rebound depolarization without spike from 14.3∓3.0 mV to 7.9∓2.0 mV (P<0.05). Nearly two-thirds of the SG neurons have rebound depolarizations modulated by HCN channel and T-type calcium channel.

Development of long-term survival spinal cord ischemia model in mice

Objective To develop long-term survival spinal cord ischemia model mimicking the injury from the surgery of thoracoabdominal aneurysm in mice.Methods Thoracic aorta was clamped for 8,10 and 12 min via left lateral thoracotomy.The mice subject to sham operation served as control group.The lumbar spinal cord blood flow was measured during thoracic aorta cross-clamping.Hind limbs movement function was evaluated before and 1 h,1,3,5,7 days,and 2,4 weeks post-injury.The slices of the spinal cord were observed and survival neurons in the ventral horn were evaluated at 7th day post-injury.Survival curve was analyzed at 4th week post-injury in mice.The data were statistically analyzed using SPSS 17.0 software.Results The blood flow was reduced to (7.2 ± 3.5) ％ of the baseline after crossclamping the thoracic aorta.There was no hind limbs movement functional deficit,and histological staining of the spinal cord ischemia slices was normal in the control group.There was a most serious hind limbs movement functional deficit at 1 st h post-injury.The duration of 8 min cross-clamping could result in movement functional deficit,but open field Basso Beattie Bresnahan (BBB) score recovered at 2nd week postinjury (P ＞ 0.05 vs.control group).The duration of 10 min cross-clamping could result in continuous and stable hind limbs movement functional deficit at 4th week (P ＜0.01 vs.control group for open field BBB score and latency to fall from Rota rod) and neuronal cell death (P ＜ 0.01 vs.control group for the ventral horn neurons).Most importantly,the mice had a long term survival in 10 min group (the median survival period was 24.7 days,P ＞0.05 vs.8 min group).The duration of 12 min cross-clamping could result in less probability of long-term survival (the median survival period was 14.7 days,P ＜0.01 vs.8 min group and 10 min group),although it could result in serious hind limbs movement functional deficit.Conclusion Ten min was the best duration for mice spinal cord ischemia model through thoracic aorta cross-clamping.This kind of model can be used to study the dynamic changes of spinal cord ischemia because of the animals' long-term survival. Key words: Spinal cord ischemia; Model,animal; Mouse; Long-term survival

Protocol for the combination of neurohistological techniques on vibratome obtained sections

The histological study of the nervous system requires the use of special techniques. Currently, no methods are available to visualize simultaneously all the cellular constituents of nervous tissue. A protocol was adapted for staining nervous tissue by modification of a formerly difficult procedure. Slices of brain and spinal cord, 4 mm thick, were taken from adult mice, previously fixed by intracardiac perfusion with 4% paraformaldehyde. Vibratome sections were obtained with thickness of 15-25 µm. These were mounted on glass slides prepared with gelatin as an adhesive. The preparations were subjected to staining protocol Luxol Fast Blue-PAS-hematoxylin (LPH) combined with silver staining method (LPH-Holmes). LPH technique yielded an excellent differentiation of gray and white matter in all regions of the nervous system. A panoramic view of the gray matter was colored pink in contrast to the myelinated nerve fibers and tracts which were light blue. The combination LPH-Holmes retained the staining characteristics but significantly improved the demarcation of axons and tracts. A protocol was standardized for the LPH and LPH-Holmes nervous tissue stains applied in combination to tissue slices obtained with a vibratome. The method was shorter, less wasteful and less expensive than the original and also better preserved the integrity of nervous tissue.

The spinal cord in vitro: What can it tell us about nociception?

The use of amphibian and mammalian in vitro spinal cord preparations, e.g., hemisected cord and transverse slices, has gained in popularity over the years due to the flexibility and ease of use of such preparations compared to classical in vivo approaches. When combined with modern experimental methodologies, such as patch clamping of visualized single cells or post-experimental neuroanatomy, this approach provides a powerful addition to the armamentarium available to study nociceptive processing in the dorsal horn. Some of these novel experimental approaches and the insight into nociception that they have provided are described below.

Synaptic blockade plays a major role in the neural disturbance of experimental spinal cord compression.

We analyzed dynamic processes of neural excitation propagation in the experimentally compressed spinal cord using a high-speed optical recording system. Transverse slices of the juvenile rat cervical spinal cord were stained with a voltage-sensitive dye (di-4-ANEPPS). Two components were identified in the depolarizing optical responses to dorsal root electrical stimulation: a fast component of short duration corresponding to pre-synaptic excitation and a slow component of long duration corresponding to post-synaptic excitation. In the directly compressed dorsal horn, the slow component was attenuated more (attenuated to 37.4 +/- 9.1% of the control) than the fast component (to 70.5 +/- 14.9%) (p < 0.01) at 400 msec after stimulation. Depolarizing optical responses to compression and to chemical synaptic blockade were similar. There was a regional difference between white matter (attenuated to 86.2 +/- 10.5%) and gray matter (to 72.6 +/- 10.4%) (p < 0.03) in compression-induced changes of the fast components; neural activity in the white matter was resistant to compression, especially in the dorsal root entry zone. Depolarizing optical signals in the region adjacent to the directly compressed site were also attenuated; the fast component was attenuated to 77.6 +/- 10.4% and the slow component to 31.8 +/- 11.3% of the control signals (p < 0.01). Spinal cord dysfunction induced by purely mechanical compression without tissue destruction was virtually restored with early decompression. We suggest that a disturbance of synaptic transmission plays an important role in the pathophysiological mechanisms of spinal cord compression, at least under in vitro experimental conditions of juvenile rats.

Postsynaptic activity of spinal motoneurons of early postnatal ratsin vitro: Effects of calcium channel blockers

Postsynaptic activity was intracellularly recorded from motoneurons in slices of the spinal cord of early postnatal rats. The amplitude of background EPSP varied within 0.4 to 4.0 mV (a mean of 2.05 ± 0.18 mV); their mean frequency was 11.5 ± 4.9 sec−1. Distributions of the EPSP amplitudes In different cells were unimodal and showed a clear right-side asymmetry. Application of calcium channel blockers (1–2 μM nifedipine, 50–100 μM Cd2+, or 500 μM amyloride) considerably modified the EPSP parameters. Nifedipine (3 cells) somewhat enhanced the amplitude of background EPSP (to 108.3 ± 6.6%, on the average) and significantly Increased their frequency (145.6 ± 14.4%). Cadmium (3 cells) and amyloride (7 cells) decreased both the EPSP amplitude (means of 88.9 ±2.8% and 73.1 ± 3.8%, respectively) and their frequency (69.0 ± 5.6% and 61.4 ± 7.8%). All tested agents evoked no dramatic shifts of the membrane potential of motoneurons. It is concluded that the observed modifications of the EPSP amplitudes and frequencies result from modulation of the activity of both pre- and post-synaptically localized high- and low-threshold Ca2+ channels. The reason for the opposite direction of nifedipine effects, as compared with those of Cd2+ and amyloride, is discussed.

Release of Ach by slices of the ventral nerve cord of the cockroach Periplaneta americana

Release of Ach by slices of the ventral nerve cord of the cockroach Periplaneta americana

Capsaicin-like effect of (6)-shogaol on substance P-containing primary afferents of rats: a possible mechanism of its analgesic action.

The effects of (6)-shogaol, a pungent component of dried ginger with a capsaicin-like chemical structure, on the release of immunoreactive substance P from the spinal dorsal horn were examined by in vitro superfusion of the dorsal-half slices of the spinal cord of the rat. (6)-Shogaol (30 microM to 1 mM) increased dose-dependently the release of immunoreactive substance P. The maximum effect of (6)-shogaol was observed at a concentration of 100 microM and less than a half of the effect of 10 microM capsaicin. The effect of (6)-shogaol (100 microM) was attenuated in slices from rats with dorsal rhizotomy and abolished by elimination of calcium ions from the perfusion medium. Pretreatment with (6)-shogaol in vitro inhibited the capsaicin-evoked release of immunoreactive substance P. On the other hand, systemic administration of (6)-shogaol (160 mg/kg) produced antinociception in rats, with a peak effect between 15 and 30 min and a smaller dose of 80 mg/kg was without effect. Treatment of rats with (6)-shogaol, at a dose of 160 mg/kg but not at 80 mg/kg, for 20 min significantly decreased release of immunoreactive substance P, evoked by capsaicin (10 microM), from the slices of cord. These data suggest that (6)-shogaol shares the sites of action with capsaicin, on the terminals of substance P-containing primary afferents, to release of the neuropeptide and inhibit the release of substance P, by subsequent stimulation of the primary afferents. The latter action of (6)-shogaol might be relevant to its analgesic effect.

Galanin receptors and their second messengers in the lumbar dorsal spinal cord.

The ligand binding properties of galanin receptors were examined in crude synaptosomal fraction preparations of lumbar dorsal spinal cord, using chloramin-T mono-iodinated porcine Tyr26 galanin as ligand. The equilibrium binding of [125I]galanin showed the presence of a single population of high-affinity binding sites with KD = 0.6 +/- 0.2 nM in a concentration of 55 +/- 15 fmol mg-1 protein (Bmax). The N-terminal fragments galanin (1-16) and galanin (1-12) fully displaced specific [125I]galanin binding from membranes with IC50 values 6 nM and 4 microM, respectively. The C-terminal fragment galanin (17-29) did not displace [125I]galanin when applied in the concentration range 10(-11)-10(-4) M. GTP inhibited the specific binding of [125I] galanin in a concentration dependent manner, with 54% inhibition at 1 mM, suggesting that the galanin receptor found in lumbar dorsal spinal cord is G-protein coupled. Second messenger systems, through which the galanin receptor in lumbar dorsal spinal cord may exert its effect, were also studied. A galanin (10 microM) produced inhibition (58%) of the depolarization induced cGMP increase was found, whereas galanin (10 microM) did not inhibit the noradrenalin (100 microM) activated cAMP synthesis or phosphoinositide turnover in tissue slices of the spinal cord. Bilateral transection of the sciatic nerve at midthigh level 14 days prior to the binding experiment was performed, a treatment which is known to cause a dramatic increase of galanin-like immunoreactivity in the superficial layers of the dorsal spinal cord, dorsal root ganglion and in galanin mRNA levels, but no significant effect on Bmax or KD of the galanin receptor was found.

Damage to the high-affinity γ-aminobutyric acid (GABA) uptake system in mouse brain by horseradish peroxidase (HRP)

Presynaptic nerve terminals when depolarized are sensitive to morphological and functional alteration by horseradish peroxidase. Mouse brain slices, 0.1 mm, depolarized by a K(+)-HEPES buffer and exposed to horseradish peroxidase exhibited alterations in both synaptic vesicle membrane structure and in high-affinity [(14)C]?-aminobutyric acid uptake. The post stimulatory retrieval of synaptic vesicles from the nerve terminal plasma membrane in the presence of horseradish peroxidase resulted in a decrease in the synaptic vesicle population with a concurrent increase in non-synaptic vesicle membrane structures. High-affinity [(14)C]?-aminobutyric acid uptake into 0.1-mm slices of mouse cerebral cortex and ponsmedulla-spinal cord was inhibited by 31% and 24%, respectively, after incubation for 60 min in K(+)-HEPES buffer containing horseradish peroxidase. Superoxide dismutase protected both the synaptic vesicle membrane and the high-affinity uptake system from the deleterious effects of horseradish peroxidase, pointing to the possible involvement of superoxide anion radicals in the horseradish peroxidase-related effects. These horseradish peroxidase induced alterations appear to be directed towards the exposed synaptic vesicle membrane, since non-stimulated brain slices exposed to horseradish peroxidase do not exhibit a reduction in either high- or low-affinity [(14)C]?-aminobutyric acid uptake. Low-affinity uptake of [(14)C]?-aminobutyric acid and [(14)C]?-aminoisobutyric acid into cortical slices was not affected after incubation in K(+)-HEPES with horseradish peroxidase. Low-affinity uptake, however, is reduced by the high-K(+)/Na(+)-free stimulatory incubation prior to uptake. It appears, thus, that high- and low-affinity uptake are distinct and different systems, with the high-affinity transport system structurally associated with synaptic vesicle membrane.

Fine structure of dividing astroglia and oligodendroglia during myelin formation in the developing mouse spinal cord.

To study the morphology and cellular relationships of dividing glial cells during myelin formation, were perfused newborn and 5-day mouse pups and embedded slices of cervical, thoracic, and lumbar cord for light and electron microscopic study. In semithin epon sections stained with toluidine blue, all levels of spinal cord at both ages contained mitotic glia in gray columns and funiculi. In electron micrographs of funiculi, dividing astroglia containing bundles of glial filaments, many glycogen granules, and had large processes extending into the surrounding neuropil. Cytoplasmic organelles of many immature interphase oligodendroglia and mitotic oligodendroblasts were similar and included microtubules, clusters of free ribosomes, and scattered profiles of granular endoplasmic reticulum. Unlike astroglia, dividing oligodendroblasts lacked large processes and in metaphase they were ellipsoids and had smooth plasma membranes. When these cells were studied in alternating serial thin and semithin sections over 10-15 micrometers distances, we did not identify connections between myelin sheaths and mitotic oligodendroblasts. Our findings indicate that oligodendroglia in developing white matter multiply before developing large processes. Our data also suggest that oligodendroglia do not divide while forming myelin.

The presynaptic effects of valproic acid in the isolated frog spinal cord

The effects of the anticonvulsant valproic acid ( n-dipropylacetate, DPA) on frog primary afferent fibers was examined with sucrose gap recordings from the dorsal roots. Addition of DPA to the superfusate consistently reduced the amplitude and duration of the dorsal root potential. In contrast, DPA augmented the depolarization of dorsal roots produced by GABA, β-alanine and taurine. It also decreased afferent fiber ‘desensitization’ to GABA. DPA depressed the ability of K + and the excitatory amino acids glutamate and aspartate to depolarize afferent fibers. In addition, the compounds decreased the amount of K + released by tetanic stimulation of the dorsal root. The K +-evoked release of tritiated GABA from cord slices was initially reduced by exposure to DPA, but was then unaffected after a longer application of the anticonvulsant. On the other hand, the high affinity uptake of tritiated GABA and glycine were almost totally blocked by the addition of DPA to the incubating medium. In sum, DPA has complex effects on neuronal membranes. Some of these effects may serve to explain the anticonvulsant actions of this drug.

γ‐Hydroxybutyric acid is not a GABA‐mimetic agent in the spinal cord

gamma-Hydroxybutyric acid (GHB), a pharmacologically active central nervous system constituent, has been postulated to function as a gamma-aminobutyric acid (GABA) agonist. This hypothesis was tested directly on GABAergic synapses in isolated, superfused frog spinal cord. Addition of GHB to the superfusate produced effects on primary afferent terminals that were distinctly different from the effects of GABA. Thus, although both compounds depressed dorsal root potentials, GHB hyperpolarized terminals while GABA depolarized the same structures. The GABA responses were antagonized by bicuculline and picrotoxin, but these alkaloids did not change GHB's actions. In addition, GHB altered neither high-affinity uptake by cord slices, nor potassium-evoked release of tritiated GABA from them. GHB did not directly release GABA from spinal slices preloaded with [3H]GABA. These observations suggest that the central nervous system actions of GHB are not dependent upon its ability to activate GABAergic synapses or to modify GABAergic mechanisms.

Pentylenetetrazol and reflex activity of isolated frog spinal cord

The superfused in vitro frog spinal cord preparation was used to investigate the effects of pentylenetetrazol (PTZ) on the spinal cord. PTZ depressed monosynaptic, but augmented polysynaptic reflexes, and decreased primary afferent deplorization. Concurrently, in Ringer's solution containing sufficient magnesium or cobalt ions to block synaptic transmission, PTZ antagonized the hyperpolarizing effects on motoneurons and the depolarizing effects on primary afferent fibers of the inhibitory amino acids GABA, beta-alanine, taurine, and glycine. PTZ did not affect responses to the excitatory amino acids glutamic acid and aspartic acid. Furthermore, PTZ did not alter high affinity uptake by cord slices, K+ -evoked release of [3H]GABA from them, or the spinal concentration of GABA. These data suggest that PTZ may produce its excitatory effects by postsynaptic blockade of inhibitory processes mediated by GABA (and possibly by other amino acids).

Effect of electrical stimulation and high potassium concentrations on the efflux of [14C] glycine from slices of spinal cord

1. The effects of electrical stimulation and solutions containing a high concentration of potassium on the efflux of [(14)C] glycine from slices of rat spinal cord have been studied.2. Slices of cord were incubated with [(14)C] glycine which rapidly accumulated in the tissue. The slices were then superfused in a small chamber and the radioactivity released from the tissue was measured. After superfusion for 60 min, 98% of the radioactivity remaining in the tissue was present as unchanged glycine.3. The spontaneous efflux of [(14)C] glycine consisted of an initial rapid phase followed by a much slower release of [(14)C] glycine. After superfusion for 60 min, more than 65% of the radioactivity taken up during the incubation period was retained by the tissue.4. When the slices were depolarized by electrical stimulation or by solutions containing a high concentration of potassium (40 mM), a striking increase in the efflux of [(14)C] glycine was produced. This effect was not reduced by the absence of calcium ions in the superfusion medium.5. Electrical stimulation produced similar increases in the efflux of [(3)H] GABA and [(14)C] glutamate from slices of cord but had no significant effects on the efflux of [(3)H] alanine or [(14)C] urea.6. The results are consistent with the suggestion that glycine may be an inhibitory synaptic transmitter substance in the mammalian spinal cord.