Alterations in AMPA receptor subunit expression after experimental spinal cord contusion injury - PubMed (original) (raw)

Alterations in AMPA receptor subunit expression after experimental spinal cord contusion injury

S D Grossman et al. J Neurosci. 1999.

Abstract

The AMPA-preferring subtype of ionotropic glutamate receptors (GluRs) is a hetero-oligomeric ion channel assembled from various combinations of four subunits: GluR1, GluR2, GluR3, and GluR4. Antagonists of these receptors can mitigate the effects of experimental spinal cord injury (SCI), indicating that these receptors play a significant role in pathophysiology after spinal trauma. We tested the hypothesis that SCI alters expression of AMPA receptors using a standardized thoracic weight-drop model of rat contusive spinal cord injury. AMPA receptor subunit expression was measured at 24 hr and at 1 month after SCI with quantitative Western blot analysis and in situ hybridization. GluR2 protein levels were preferentially reduced near the injury site 24 hr after SCI. This reduction persisted at 1 month. At a cellular level, a significant decrease in both GluR2 and GluR4 mRNA was found in spared ventral motor neurons adjacent to the injury site and distal to it, with other AMPA subunit mRNAs maintained at control levels. In contrast, only GluR1 mRNA was decreased in the sympathetic preganglionic neurons of the intermediolateral horn. These results suggest population-specific and long-lasting changes in neuronal AMPA receptor composition, which may alter response to glutamate after SCI. These alterations may contribute not only to acute neuropathological consequences of injury, but they may also be partially responsible for the altered functional state of preserved tissue seen chronically after SCI.

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Figures

Fig. 1.

Fig. 1.

Outcome of the experimental weight-drop model of contusive spinal cord injury used in the study. A, Results of behavioral testing of rats using the combined behavioral score (CBS Score) (Noble and Wrathall, 1985), a measure of overall hindlimb deficit. B, Results using the Basso, Beattie, Bresnahan scale (BBB Score) (Basso et al., 1995) of hindlimb locomotor ability (see Materials and Methods for full description). Both measures of hindlimb function demonstrate profound deficit at 24 hr and partial recovery with a residual chronic deficit at 1 month. Data points indicate mean ± SD for the groups of rats (n = 28 each) after T8 laminectomy (○—○) or laminectomy plus SCI rats (●—●) produced with a weight-drop device (10 gm × 2.5 cm). Because of the reproducibility of the injury model, the SD bars are generally hidden by the symbols.C, Tracings of a representative injured spinal cord at 1 month after injury showing the epicenter (region of maximal damage) and sections 1 and 4 mm rostral and caudal to it.Black region indicates the lesion area, which consists of cavities and a loose accumulation of lesion cells, primarily macrophages (Noble and Wrathall, 1985, 1989; Wrathall et al., 1998).

Fig. 2.

Fig. 2.

Levels of AMPA receptor subunit proteins are altered at 24 hr and 1 month after SCI, as measured by quantitative analysis of Western blots. A, Typical Western blots using polyclonal antibodies directed against AMPA receptor subunits are shown. The top blot on each column represents control protein levels, the middle blot represents acute SCI levels at 24 hr after injury, and the bottom blot_represents levels at 1 month after SCI for GluR1, GluR2, GluR2/3, GluR4, and NSE. C, Cervical; T, thoracic;L, lumbar. B, Relative protein levels were quantified for each subunit as described in Materials and Methods for GluR1, GluR2, GluR2/3, GluR4, and NSE. For all graphs, white bar = control levels (n = 16–18 rats), cross-hatched bar = 24 hr after SCI (n = 8), black bar = 1 month after SCI (n = 9). Repeat-measures ANOVA was used to compare all nine experimental groups for each antibody with_post hoc comparisons by the Tukey test. *Significantly different from level in laminectomy controls, p ≤ 0.05.

Fig. 3.

Fig. 3.

Dark-field microscopy showing autoradiographic grains overlying areas of mRNA expression in thoracic spinal cord hemisections from laminectomy control and acute SCI rats.A, Nissl-stained hemisection of normal spinal cord.B, Normal expression of GluR1 mRNA. C, Reduced GluR1 mRNA expression in a section 2 mm caudal to the epicenter 24 hr after SCI. D, Normal expression of GluR3 mRNA.E, Tracing of normal spinal cord showing regions examined in this study. F, Normal expression of GluR2 mRNA. G, Decreased expression of GluR2 mRNA 2 mm caudal to the epicenter 24 hr after SCI. H, Normal expression of GluR4 mRNA. Exposure times and the specific activities of the probes were similar for all sections. Scale bar, 0.5 mm.

Fig. 4.

Fig. 4.

Expression of AMPA subunit GluR2 mRNA is specifically altered in ventral motor neurons 1 month after SCI. Bright-field photomicrographs showing healthy appearing, Nissl-stained, ventral motor neurons. VMNs are located 4 mm caudal to the thoracic epicenter in controls and SCI rats, at 1 month after laminectomy or weight-drop injury. A, GluR2 mRNA in VMNs of control rats is moderately expressed. B, Decreased GluR2 mRNA in VMNs from injured rats. C, GluR4 mRNA expression is high in control VMNs. D, VMNs from SCI rats express slightly but significantly less GluR4 mRNA than controls. E, GluR1 mRNA is sparse in VMNs from control spinal cord.F, VMNs from SCI rats express similar levels of GluR1 mRNA as controls. G, GluR3 mRNA expression is high in control spinal cord and is similarly high 1 month after injury (H). Scale bar = 5 μm.

Fig. 5.

Fig. 5.

Different populations of neurons exhibit distinct alterations in AMPA receptor subunit mRNA expression after SCI.Graphs represent grain density overlying individual ventral motor neurons (VMN) or IML neurons expressed as a percentage of grain counts from adjacent laminectomy control sections on the same slide. Sections analyzed were from 4 mm rostral and caudal to the epicenter of injury, where for the most part tissue was morphologically intact. A, IML neurons at 24 hr after SCI. B, Ventral motor neurons at 24 hr after SCI. C, IML neurons at 1 month after SCI.D, Ventral motor neurons at 1 month after SCI.n = 5 rats per group with 93–141 VMNs and IML neurons analyzed per animal. *Significant difference between grains per square micrometer of neuron in control and SCI groups (Student’s_t_ test, p ≤ 0.05).

Fig. 6.

Fig. 6.

Quantitative analysis shows that GluR2 mRNA is chronically downregulated in ventral motor neurons adjacent to the injury site and also distally in the lumbar enlargement.Graph represents grain counts as a percentage of control (_y_-axis) for individual ventral motor neurons at various millimeter distance increments rostral and caudal to the injury epicenter (_x_-axis). There is a significant decrease in GluR2 mRNA grain density in neurons counted at each position compared with neurons on control sections from that position on the same slide (Student’s t test, p ≤ 0.05). There is also a significant effect of distance from the epicenter on the relative decrease of GluR2 mRNA (repeat-measures ANOVA,p = 0.001). The _dotted lines_indicate statistically similar effects at ±2, ±4, ±8 mm, and −15 to −20 mm, whereas between the points indicated by the lines the relative levels of GluR2 mRNA are significantly different (Tukey post hoc, p ≤ 0.05).

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